WO2011069586A1 - Solution contenant du mannose pour la lyophilisation, la transfection et/ou l'injection d'acides nucléiques - Google Patents

Solution contenant du mannose pour la lyophilisation, la transfection et/ou l'injection d'acides nucléiques Download PDF

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WO2011069586A1
WO2011069586A1 PCT/EP2010/006788 EP2010006788W WO2011069586A1 WO 2011069586 A1 WO2011069586 A1 WO 2011069586A1 EP 2010006788 W EP2010006788 W EP 2010006788W WO 2011069586 A1 WO2011069586 A1 WO 2011069586A1
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mage
nucleic acid
sequence
lyophilization
transfection
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PCT/EP2010/006788
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Thorsten Mutzke
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Curevac Gmbh
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Priority to EP10779700.3A priority Critical patent/EP2510100B1/fr
Priority to US13/509,564 priority patent/US20120258046A1/en
Publication of WO2011069586A1 publication Critical patent/WO2011069586A1/fr
Priority to US14/492,334 priority patent/US9616084B2/en
Priority to US15/451,675 priority patent/US20170182081A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the present invention is directed to (the use of) a solution containing at least one nucleic acid (sequence) and free mannose for lyophilization, transfection and/or injection, particularly of RNA and mRNA.
  • the inventive solution exhibits a positive effect on stabilization of the nucleic acid (sequence) during lyophilization and storage but also leads to a considerable increase of the transfection efficiency of a nucleic acid. It thus also increases in vivo expression of a protein encoded by such a nucleic acid upon increased transfection rate.
  • the present invention is furthermore directed to a method of lyophilization using the mannose-containing solution, to pharmaceutical compositions, vaccines, kits, first and second medical uses applying such a mannose-containing solution and/or a nucleic acid (sequence) lyophilized or resuspended with such a solution.
  • nucleic acids In gene therapy and many other therapeutically relevant biochemical and biotechnological applications the use of nucleic acids for therapeutic and diagnostic purposes is of major importance. As an example, rapid progress has occurred in recent years in the field of gene therapy and promising results have been achieved. Nucleic acids are therefore regarded as important tools for gene therapy and prophylactic and therapeutic vaccination against infectious and malignant diseases. Nucleic acids, both DNA and RNA, have been used widely in gene therapy, either in naked or in complexed form. In this context, the application of nucleic acids and particularly of RNA for therapeutic vaccination is revised permanently. On the one hand, nucleic acids and particularly RNA or mRNA molecules can be optimized for a more efficient transcription rate.
  • the 5' Cap structure, the untranslated and translated regions are typically modified to stabilize the molecule or to change its characteristics to enhance its translation properties (see e.g. Pascolo, S. (2008), Handb Exp Pharmacol (1 83): 221 -35).
  • different formulations of nucleic acids and particularly of mRNA molecules or different delivery routes are investigated to achieve improved expression levels.
  • the encapsulation into cationic liposomes or cationic polymers see e.g. Hoerr, I., R. Obst, eta/. (2000), Eur J Immunol 30(1 ): 1 -7. ; Hess, P. R., D. Boczkowski, et a/.
  • RNA pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 184(2): 465-72; Boczkowski, et a/, 1996, supra), and the direct injection of naked RNA (see Hoerrr et a/ 2000, supra).
  • RNA thus represents a favored tool in modern molecular medicine. It also exhibits some superior properties over DNA cell transfection. As generally known, transfection of DNA molecules may lead to serious problems. E.g. application of DNA molecules bears the risk that the DNA integrates into the host genome.
  • RNA particularly mRNA
  • An advantage of using RNA rather than DNA is that no virus-derived promoter element has to be administered in vivo and no integration into the genome may occur.
  • RNA has not to overcome the barrier to the nucleus.
  • a main disadvantage resulting from the use of RNA is due to its huge instability.
  • DNA e.g., naked DNA
  • DNA introduced into a patient' circulatory system is typically not stable and therefore may have little chance of affecting most disease processes (see e.g. Poxon et a/., Pharmaceutical development and Technology, 5(1 ), 1 15-122 (2000)) the problem of stability is even more evident in the case of RNA.
  • the physico chemical stability of RNAs in solution is extremely low. RNA is very susceptible to hydrolysis by ubiquitous ribonucleases and is typically completely degraded already after a few hours or days in solution. This even occurs in the absence of RNases, e.g. when stored a few hours or days in solution at room temperature.
  • RNA is typically stored at -20°C or even -80°C and RNAse free conditions to prevent a prior degradation of the RNA.
  • This method does not prevent a loss of function effectively and additionally is very cost-intensive for shipping when these temperatures have to be guaranteed.
  • One further method for stabilization comprises lyophilization or freeze-drying of the RNA. Lyophilization is a worldwide known and recognized method in the art to enhance storage stability of temperature sensitive biomolecules, such as nucleic acids. During lyophilization, typically water is removed from a frozen sample containing nucleic acids via sublimation. The process of lyophilization is usually characterized by a primary and a secondary drying step. During the primary drying step, free, i.e.
  • the sample containing nucleic acids is initially cooled below the freezing point of the solution and accordingly of the water contained therein. As a result, the water freezes. Dependent on temperature, rate of cooling down (freezing rate), and the time for freezing, the crystal structure of water is changed. This exhibits physical stress on the nucleic acid (sequence) and other components of the solution, which may lead to a damage of the nucleic acid, e.g. breakage of strands, loss of supercoiling, etc. Furthermore, due to the decrease of volume and loss of the hydration sphere, autocatalytic degradation processes are favored e.g. by traces of transition metals. Additionally, significant changes of pH are possible by concentration of traces of acids and bases.
  • Lyophilization involves two stresses, freezing and drying. Both are known to damage nucleic acids, such as non-viral vectors or plasmid DNA. In the literature, a number of cryoprotectants and lyoprotectants are discussed for lyophilization purposes to prevent these damages. In this context, cryoprotectants are understood as excipients, which allow influencing the structure of the ice and/or the eutectical temperature of the mixture. Lyoprotectants are typically excipients, which partially or totally replace the hydration sphere around a molecule and thus prevent catalytic and hydrolytic processes.
  • lyophilization causes the removal of the hydration sphere around the DNA, wherein it appears that there are approximately 20 water molecules per nucleotide pair bound most tightly to DNA. These water molecules do not form an ice-like structure upon low-temperature cooling. Upon DNA dehydration over hygroscopic salts at 0% relative humidity, only five or six water molecules remain (see e.g. Tao et al, Biopolymers, 28, 1019-1030 (1 989)). Lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure, breaks of the nucleic acid chain(s) or the concentration of reactive elements such as contaminating metals.
  • Lyophilization can also cause damage upon the initial lyophilization process in other nucleic acid, e.g. RNA.
  • Agents that can substitute for non-freezable water, such as some carbohydrates, can demonstrate cryoprotective properties for DNA and other molecules during lyophilization of intact bacteria (see e.g. Israeli et al, Cryobiology, 30, 51 9-523 (1993); or Rudolph et al, Arch. Biochem. Biophys., 245, 134-143 (1986)).
  • specific carbohydrates are utilized in the art as lyoprotective substances for enhancing stability of the nucleic acid (sequence) during lyophilization. They exhibit an effect on storage stability after lyophilisation of pure nucleic acids or nucleic acid (sequence) complexes (see e.g. Maitani, Y., Y. Aso, et al. (2008), Int J Pharm 356(1 -2): 69- 75 ; Quaak, S. G., J. H. van den Berg, et al. (2008), Eur J Pharm Biopharm 70(2): 429-38 ; Jones, K. L., D. Drane, et al.
  • Lyoprotective properties are particularly described for sucrose, glucose, and trehalose. They allow to restore at least in part the transfection efficiency which is otherwise lost in many cases after lyophilisation (see Maitani et al, 2008, supra; Yadava, P., M. Gibbs, et al. (2008). AAPS PharmSciTech 9(2): 335-41 ; Werth, S., B.
  • Sugars are able to prevent loss in activity due to the lyophilization process mainly by preventing particle fusion/aggregation especially in the case of liposome complexed nucleic acids (see Yadava et al, 2008, supra; Katas, H., S. Chen, etal. (2008), J Microencapsul: 1 -8; Molina etal, supra, 2001 ).
  • Poxon et al. (2000, supra) investigated the effect of lyophilization on plasmid DNA activity.
  • Poxon et al. (2000, supra) hypothetized, that a change in the DNA conformation from supercoiled to open circular and linear form would be indicative of damage of the plasmid DNA.
  • the percentage of supercoiled DNA did not change after lyophilization and subsequent DMED treatment, suggesting that other effects drew responsible for the loss of transfection efficiency.
  • Li et al. (see Li, B., S. Li, et al. (2000), J Pharm Sci 89(3): 355-64) furthermore showed that disaccharides are superior to monosaccharides using them as a cryoprotectant for lyophilization of lipid based gene delivery systems due to the prevention of aggregation. They noted that it is very important to prevent the particle size of the complexes during lyophilization. Unfortunately, in a specific example of lipid based gene delivery systems, lyophilization with mannose led to an increase in particle size, which was regarded as negative for transfection efficiency. Additionally Li et al. (2000, supra) showed that lipid delivery systems can be stored at room temperature without loss of transfection efficiency when lyophilized in 10% sucrose.
  • Li et al. (2000, supra) did not examine the stabilization due to the presence of mannose as a lyoprotectant. More importantly, they did not observe an increase in the expression of the encoded protein due to the presence of sugar (sucrose and trehalose) in the injection buffer.
  • Jones et a/ (2007, supra) is one rare document, which examines the effect of sugars on long term stability of mRNA. It describes the possibility to prevent storage depending loss of transfection activity in vitro. Jones et al (2007, supra) uses trehalose as a lyoprotectant and shows a preventive effect on the loss of transfection activity at a storage temperature of 4°C for a period of 6 months. Integrity of the mRNA was only measured by loss of weight after recovering. At elevated temperatures (room temperature and higher) degradation and a dramatic loss of transfection efficiency took place. Additionally; transfection efficiency could not be improved using trehalose as lyoprotectant.
  • specific carbohydrates may also be utilized to improve biological activity and/or transfection efficiency, which is, at least at a first glance, independent from stability issues.
  • specific carbohydrates e.g. of mannose may be attributed to the interaction of these carbohydrates with specific receptors in the cell.
  • mannose may involve the mannose receptor targeted transfer.
  • the mannose receptor (MR) is primarily present on dendritic cells (DCs) and macrophages.
  • the carbohydrate recognition domains of the MR recognizes carbohydrates (e.g. mannose, fucose, glucose, N-Acetylglucosamine, maltose) on the cell walls of infectious agents (mainly bacteria and yeast) which leads to rapid internalization and phagocytosis.
  • mannose was covalently bound to the vector to ensure a combined uptake due to binding to the mannose receptor.
  • the expression of the mannose receptor is restricted to a few cell types (especially dendritic cells) which are not excessively present in the dermis and therefore it appeared unlikely that free mannose improves the expression of the encoded protein due to an increased uptake in mannose receptor expressing cells.
  • sucrose is a common trigger for endocytosis in animal cells and therefore the ODN internalizes into endosomes together with the sucrose.
  • Sun et al. (2007) only examined in vitro transfection assays which are very difficult to transfer to the in vivo situation due to the dilution effect. In tissues it thus appeared very unlikely that the nucleic acid and the sugar molecule enter the cell at the same time.
  • RNA is very cost-intensive to ensure temperatures at -20°C and below during shipment.
  • the problem underlying the present invention is solved by (the use of) a solution containing at least one nucleic acid (sequence) and free mannose for lyophilization, transfection and/or injection.
  • the inventive solution containing at least one nucleic acid (sequence) and mannose stabilizes the at least one nucleic acid (sequence) contained in the inventive solution during lyophilization and/or improves biological activity of the nucleic acid (sequence). This is particularly preferable true, if a protein is encoded by the at least one nucleic acid (sequence), as expression of an encoded protein may be increased thereby.
  • the present invention thus provides (the use of) a solution containing at least one nucleic acid (sequence) and (free) mannose for lyophilization, transfection and/or injection.
  • free mannose is preferably understood as a mannose, which is not covalently bound and/or conjugated, preferably not covalently bound and/or conjugated to the nucleic acid (sequence) to be lyophilized, transfected and/or injected.
  • "Free" mannose may therefore comprise a free, non-covalently bound and/or unconjugated mannose, preferably with repsect to the nucleic acid (sequence) to be lyophilized, transfected and/or injected.
  • mannose is preferably a sugar monomer of the aldohexose series of carbohydrates.
  • Mannose as defined herein typically has the molecular formula C 6 H 12 C 6 , is also known under its lUPAC nomenclature as (25,35,4 ⁇ ,5 ⁇ - Pentahydroxyhexanal, (2A',3A , ,45,5i)-Pentahydroxyhexanal. It is preferably identified under CAS number 31 103-86-3 and typically exhibits the following general structure:
  • Mannose is typically formed by the oxidation of mannitol. It can also be formed from D- glucose in the Lobry-de Bruyn-van Ekenstein transformation. Mannose as defined herein typically occurs in two diastereomeric isoforms, D-Mannose and L-Mannose (CAS numbers 3458-28-4 for D-mannose and 10030-80-5 for L-mannose). D-mannose is sold as a naturopathic remedy for urinary tract infections, and it is claimed to work through the disruption of adherence of bacteria in the urinary tract. D-Mannose and L-Mannose can be illustrated as the D and L straight-chain forms of mannose using Fischer projections according to the following structures:
  • mannose as used herein is a D-Mannose.
  • D- Mannose may be depicted according to at least one of the D-Mannose isomers a-D- Mannofuranose, ⁇ -D-Mannofuranose, a-D-Mannopyranose and ⁇ -D-Mannopyranose as represented by following Haworth-structures:
  • D- Mannose forms anomers, wherein a-D-Mannofuranose ocurrs in a concentration/frequency of less than 1 %, ⁇ -D-Mannofuranose in a concentration/frequency of less than 1 %, a-D- Mannopyranose in a concentration/frequency of about 67 % and ⁇ -D-Mannopyranose in a concentration/frequency of about 33 %.
  • D-Mannose may be selected more preferably from at least one, two, three or four of the anomers a-D-Mannofuranose, ⁇ -D- Mannofuranose, ⁇ -D-Mannopyranose and/or ⁇ -D-Mannopyranose. Most preferably, upon solubilization in an aqueous solution mannose typically forms the above anomers in an equilibrity reaction, typically in the above concentrations.
  • mannose as used herein is selected from an anomeric mixture of D-Mannose, preferably an anomeric mixture comprising a-D- Mannofuranose, ⁇ -D-Mannofuranose, ⁇ -D-Mannopyranose and ⁇ -D-Mannopyranose, more preferably in the above concentrations/frequencies.
  • mannose as used herein may be selected from L-mannose or a racemic mixture of D- Mannose and/or L-Mannose, wherein D-mannose preferably as described above.
  • Such mixtures may be obtained e.g. by a non-selective synthesis of mannose, e.g. by nonselective oxidation of mannitol.
  • An anomeric mixture may furthermore be obtained by solubilization of mannose in an aqueous solution, e.g. in water, WFI, or any buffer or solution as defined herein.
  • mannose as used herein is typically present in the inventive solution for lyophilization, transfection and/or injection in a concentration of about 0.01 to about 10% (w/w), preferably in a concentration of about 0.01 to about 10% (w/w), more preferably in a concentration of about 0.1 to about 7.5% (w/w), even more preferably in a concentration of about 0.5 to about 5% (w/w), and most preferably in a concentration of about 1 to about 4% (w/w), e.g. a concentration of about 2 to about 4% (w/w), such as about 2.5 % (w/w).
  • a concentration of about 1 % (w/w) mannose corresponds to a concentration of about 55,506 mM mannose. Any of the above and herein mentioned values and concentrations for mannose in % (w/w) may thus be calculated in mM on the above basis.
  • the present invention provides (use of) a solution containing at least one nucleic acid sequence and free mannose for lyophilization, transfection and/or injection of the at least one nucleic acid (sequence). Lyophilization, transfection and/or injection may be carried out in vivo, in vitro or ex vivo.
  • a lyophilized nucleic acid (sequence) may be any suitable nucleic acid, selected e.g. from any (double-stranded or single-stranded) DNA, preferably, without being limited thereto, e.g.
  • genomic DNA single-stranded DNA molecules, double- stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, a (short) DNA oligonucleotide ((short) oligodesoxyribonucleotides), or may be selected e.g. from any PNA (peptide nucleic acid) or may be selected e.g.
  • RNA from any (double- stranded or single-stranded) RNA, preferably, without being limited thereto, a (short) RNA oligonucleotide ((short) oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, an antisense RNA, a micro RNA or riboswitches, ribozymes or aptamers; etc.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • vRNA viral RNA
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is an RNA. More preferably, the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a (linear) single-stranded RNA, even more preferably an mRNA.
  • 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 nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a single- or a double-stranded nucleic acid (molecule) (which may also be regarded as a nucleic acid (molecule) due to non-covalent association of two single-stranded nucleic acid(s) (molecules)) 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 acid molecules are typically formed by a longer and a shorter single-stranded nucleic acid molecule or by two single stranded nucleic acid molecules, which are about equal in length, wherein one single- stranded nucleic acid molecule is in part complementary to the other single-stranded nucleic acid molecules molecule and both thus form a double-stranded nucleic acid molecules molecule in this region, i.e.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a single-stranded nucleic acid molecule.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a coding nucleic acid, e.g. a DNA or RNA.
  • a coding DNA or RNA may be any DNA or RNA as defined above.
  • such a coding DNA or RNA may be a single- or a double-stranded DNA or RNA, more preferably a single-stranded DNA or RNA, and/or a circular or linear DNA or RNA, more preferably a linear DNA or RNA.
  • the coding DNA or RNA may be a (linear) single-stranded DNA or RNA.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a ((linear) single- stranded) messenger RNA (mRNA).
  • mRNA messenger RNA
  • Such 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 nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may 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.
  • telomeres selected from pathogenic proteins as defined herein 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 protein or peptide suitable for a specific (therapeutic) application, wherein the coding DNA or RNA 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 as defined herein 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 protein or peptide suitable for a specific (therapeutic) application, wherein the
  • therapeutically active proteins may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection. These may be selected from any naturally occurring recombinant or isolated protein known to a skilled person from the prior art. Without being restricted thereto 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.
  • CCCC positionally conserved cysteine residues
  • WSXWS conserved sequence motif Trp-Ser-X-Trp-Ser
  • 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 IL-2- subfamily, e.g. IL-2, IL-4, IL-7, IL-9, IL-15, etc., or the cytokines IL-1 alpha, IL-1 beta, IL-10 etc.
  • GM-CSF subfamily e.g. IL-3, IL-5, GM-CSF
  • the IL-6-subfamily e.g. IL-6, IL-1 1 , IL-12
  • the IL-2- subfamily e.g. IL-2, IL-4, IL-7, IL-9, IL-15, etc.
  • Therapeutically active proteins may also comprise cytokines of class II 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 II 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., or cytokines of the family of chemokines, which comprise 7 transmembrane helices and interact with G-protein, e.g.
  • Therapeutically active proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be selected from any of the proteins given in the following: 0ATL3, 0FC3, 0PA3, 0PD2, 4- 1BBL, 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, AC A DM, ACADS, ACADVL, ACAT1, ACCPN
  • Therapeutically active proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • 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.
  • a therapeutically active protein which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • an innate immune response comprises activation of a pattern recognition receptor, such as e.g. a receptor selected from the Toll-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 Toll-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 human proteins involved in adjuvant effects may be used as well.
  • Human adjuvant proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection typically comprise any human 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 TLR.
  • human adjuvant proteins encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be 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-1 , 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 INH, C4bp, MCP, D
  • NF- ⁇ , C-FOS, c-Jun, c-Myc e.g. IL-1 alpha, IL-1 beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFN beta; from costimulatory molecules, including CD28 or CD40-ligand or PD1 ; protein domains, including LAMP; cell surface proteins; or 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.
  • costimulatory molecules including CD28 or CD40-ligand or PD1 ; protein domains, including LAMP; cell surface proteins; or human adjuvant proteins including CD80, CD81 , CD86, trif, flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any species homolog of any
  • Pathogenic adjuvant proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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, protozoan 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 preferably from pathogenic adjuvant
  • bacterial (adjuvant) proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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, Hsp100; OmpA (Outer membrane protein) from gram-negative bacteria; bacterial porins, including OmpF; bacterial toxins, including pertussis toxin (PT) from Bordetella 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-labile entero
  • CT
  • Bacterial (adjuvant) proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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, Clostridium, Escherichia, Helicobacter, Lachnospiraceae, Legionella, Listeria, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Roseburia, Salmonella, Serpulina, Serratia, Shigella, Treponema, Vibrio, Wolinella, Yersinia, more preferably flagellins from the species, without being limited thereto
  • Bacterial flagellins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection even more preferably comprise a sequence selected from the group comprising any of the following sequences as referred to their accession numbers: organism species gene name accession No CI No
  • Protozoan proteins which may also be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be selected from any protozoan protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoan heat shock proteins, LeIF from Leishmania spp, profilin- like protein from Toxoplasma gondii, etc.
  • Viral proteins which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection may finally be selected from any further pathogenic protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Keyhole limpet hemocyanin (KLH), OspA, etc.
  • KLH Keyhole limpet hemocyanin
  • OspA OspA
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 naive antigen-specific T cells by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive 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 II molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. By 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.
  • T cells differ in the class of MHC molecule that they recognize.
  • MHC class I and MHC class II- differ in their structure and expression pattern on tissues of the body.
  • CD4 + T cells bind to the MHC class II molecule and CD8 + T cells to the MHC class I molecule.
  • MHC class I and MHC class II 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. Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next.
  • 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 II 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 II molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class II molecules bind peptides from proteins which are degraded in endosomes.
  • TH2 cel ls 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.
  • Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of TH1 cells, whereas extracellular antigens tend to stimulate the production of TH2 cel ls.
  • 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 naive B cells to secrete IgM, and induce the production of weakly opsonising antibodes such as lgG1 and lgG3 (mouse) and lgG2 and lgG4 (human) as well as IgA and IgE (mouse and human).
  • antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection typically comprise any antigen, falling under the above definition, more preferably protein and peptide antigens, e.g. tumor antigens, allergy antigens, auto-immune self- antigens, pathogens, etc.
  • antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • Allergy antigens are typically antigens, which cause an allergy in a human and may be derived from either a human or other sources.
  • Antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • host organism e.g. a human
  • tumor antigens e.g. tumor antigens, self-antigens or auto-antigens, such as auto-immune self-antigens, etc.
  • 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.
  • 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) degenerated 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) and tumor-associated- antigens (TAAs). TSAs can only be presented by tumor cells and never by normal "healthy" cells.
  • 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 as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection are shown in Tables 1 and 2 below. These tables illustrate specific (protein) antigens (i.e. "tumor antigens") with respect to the cancer disease, they are associated with. According to the invention, the terms “cancer diseases” and “tumor diseases” are used synonymously herein. Table 1 : Antigens expressed in cancer diseases
  • colorectal cancer gastric cancer
  • lung cancer head and neck cancer
  • leukemia esophageal cancer
  • gastric cancer cervical cancer
  • ovarian adenocarcinoma antigen cancer breast cancer
  • bladder cancer head and neck cancer, lung cancer, melanoma,
  • gastric cancer pancreatic cancer
  • liver cancer breast cancer
  • gallbladder cancer colon cancer
  • ovarian cancer colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, uterus cancer, cervix carcinoma, colon cancer,
  • gut carcinoma colorectal cancer, colon cancer, hepatocellular cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, liver cancer cervix cancer, bladder
  • bladder cancer lung cancer, T-cell cyp-B cyclophilin B leukemia, squamous cell carcinoma,
  • DAM-10/MAGE- differentiation antigen melanoma melanoma skin tumors, ovarian
  • DAM-6/MAGE- differentiation antigen melanoma melanoma skin tumors, ovarian
  • lung cancer ovarian cancer, head and neck cancer, colon cancer
  • EGFR/Her1 pancreatic cancer breast cancer lung cancer, breast cancer, bladder tumor cell-associated extracellular cancer, ovarian cancer, brain
  • EpCam epithelial cell adhesion molecule cancer EpCam epithelial cell adhesion molecule cancer, lung cancer
  • EZH2 (enhancer of Zeste homolog 2) prostate cancer, breast cancer
  • Fra-1 Fos-related antigen-1 renal cell carcinoma thyroid cancer leukemia, renal cell carcinoma, head and neck cancer, colon cancer,
  • G250/CAIX glycoprotein 250 ovarian cancer, cervical cancer bladder cancer, lung cancer,
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • breast cancer bladder cancer, human epidermal receptor- melanoma, ovarian cancer, pancreas
  • Her2/neu/ErbB2 2/neurological cancer gastric cancer
  • breast cancer melanoma
  • lung cancer ovarian cancer
  • sarcoma human telomerase reverse Non-Hodgkin-lymphoma
  • acute hTERT transcriptase leukemia acute hTERT transcriptase leukemia
  • IL-13Ra2 interleukin 13 receptor alpha 2 glioblastoma chain
  • tongue cancer hepatocellular carcinomas, melanoma, gastric cancer, esophageal, colon cancer,
  • bladder cancer bladder cancer, head and neck cancer, melanoma, colon cancer,
  • bladder cancer bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A2 melanoma antigen-A2 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A4 melanoma antigen-A4 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A6 melanoma antigen-A6 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • small cell cancer of lung small cell cancer of lung, neuroblastoma, Wilm' tumor, melanoma, thyroid cancer, kidney cancer, testicle cancer, pancreas
  • bladder cancer head and neck cancer, melanoma, sarcoma, B- lymphoma, hepatoma, pancreatic cancer, ovarian cancer, breast
  • gastric cancer colon cancer
  • lung cancer breast cancer
  • breast cancer ovarian
  • colorectal cancer gastric cancer
  • the tumor antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection are selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1 , 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-catenin/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, CD
  • the tumor antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection are selected from the group consisting of MAGE-A1 (e.g. MAGE-A1 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 1 730
  • WT1 e.g. WT1 according to accession number NM_00037
  • PRAME e.g. PRAME according to accession number NM_0061 15
  • EGFRI epidermal growth factor receptor 1
  • EGFRI epidermal growth factor receptor 1
  • 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_1 98253
  • 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
  • PSMA etc.
  • the tumor antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may form a cocktail of antigens, e.g. in an active (immunostimulatory) composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of prostate cancer (PCa), preferably of neoadjuvant and/or hormone-refractory prostate cancers, and diseases or disorders related thereto.
  • a cocktail of antigens e.g. in an active (immunostimulatory) composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of prostate cancer (PCa), preferably of neoadjuvant and/or hormone-refractory prostate cancers, and diseases or disorders related thereto.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three or four (preferably different) antigens of the following combinations of antigens:
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three or four (preferably different) antigens:
  • At least one antigen is selected from:
  • ⁇ STEAP Symetic Epithelial Antigen of the Prostate
  • further antigen(s) is (are) selected from at least one antigen of any of the following specific antigens or combinations thereof: ⁇ PSA (Prostate-Specific Antigen), or
  • PSMA Prostate-Specific Membrane Antigen
  • PSCA Prostate Stem Cell Antigen
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be at least one RNA, more preferably at least one mRNA, encoding four (preferably different) antigens selected from PSA, PSMA, PSCA and STEAP.
  • the tumor antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may form a cocktail of antigens, e.g.
  • NSCLC non- small cell lung cancers
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following group of antigens:
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three, five or six (preferably different) antigens of the following combinations of antigens:
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following combinations of antigens:
  • VVT1 , 5T4 and NY-ESO-1 • VVT1 , 5T4 and NY-ESO-1 , or
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also be at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens exclusively selected from any of the antigens of the above mentioned group(s) or subgroup(s) comprising (at least) any one of the following combinations of antigens:
  • VVT1 VVT1 , 5T4 and Survivin, or
  • VVT1 VVT1 , NY-ESO-1 and Survivin, or
  • VVT1 VVT1 , Survivin and MAGE-C2, or
  • the tumor antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may form a cocktail of antigens, e.g. in an active (immunostimulatory) composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of non- small cell lung cancers (NSCLC), preferably selected from the three main sub-types squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma, or of disorders related thereto.
  • NSCLC non- small cell lung cancers
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is preferably at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens, a) wherein at least one, preferably at least two, three, four, five or even six, of these at least two antigens is (are) selected from:
  • the further antigen(s) is (are) selected from at least one antigen as defined herein, preferably in any of the herein mentioned combinations, groups or subgroups of antigens, e.g. the further antigen(s) is (are) selected from, e.g.:
  • the at least one antigen(s) according to a) is (are) selected from:
  • the at least one antigen(s) according to b) is (are) selected from an antigen (antigens) as defined in one of the following combinations:
  • VVT1 and MAGE-C1 VVT1 and MAGE-C1 ;
  • HER-2/NEU and NY-ESO-1 HER-2/NEU and CEA; or
  • HER-2/NEU and Survivin or HER-2/NEU and MAGE-C1 ; or HER-2/NEU and MAGE-C2; or NY-ESO-1 and CEA; or
  • NY-ESO-1 and Survivin or NY-ESO-1 and MAGE-C1 ; or NY-ESO-1 and MAGE-C2; or CEA and Survivin; or
  • VVT1 VVT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or
  • VVT1 VVT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 and Survivin; or
  • VVT1 VVT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C1 ;
  • WT1 WT1, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2; or
  • the at least one antigen(s) according to b) is (are) selected from the following combination:
  • each of the at least two (preferably different) antigens as defined herein may be encoded by one (monocistronic) RNA, preferably one (monocistronic) mRNA.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may comprise at least two (monocistronic) RNAs, preferably mRNAs, wherein each of these at least two (monocistronic) RNAs, preferably mRNAs, may encode just one (preferably different) antigen, preferably selected from one of the above mentioned combinations.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may comprise (at least) one bi- or even multicistronic RNA, preferably mRNA, i.e. (at least) one RNA which carries two or even more of the coding sequences of at the least two (preferably different) antigens, preferably selected from one of the above mentioned combinations.
  • Such coding sequences of the at least two (preferably different) antigens of the (at least) one bi- or even multicistronic RNA may be separated by at least one IRES (internal ribosomal entry site) sequence, as defined below.
  • the term "encoding at least two (preferably different) antigens” may mean, without being limited thereto, that the (at least) one (bi- or even multicistronic) RNA, preferably a mRNA, may encode e.g. at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve (preferably different) antigens of the above mentioned group(s) of antigens or their fragments or variants. More preferably, without being limited thereto, the (at least) one (bi- or even multicistronic) RNA, preferably mRNA, may encode e.g.
  • IRES internal ribosomal entry site
  • IRES sequences can function as a sole ribosome binding site, but it can also serve to provide a bi- or even multicistronic RNA as defined above which codes for several proteins, which are to be translated by the ribosomes independently of one another.
  • 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection may comprise a mixture of at least one monocistronic RNA, preferably mRNA, as defined above, and at least one bi- or even multicistronic RNA, preferably mRNA, as defined above.
  • the at least one monocistronic RNA and/or the at least one bi- or even multicistronic RNA preferably encode different antigens or their fragments or variants, the antigens preferably being selected from one of the above mentioned groups or subgroups of antigens, more preferably in one of the above mentioned combinations.
  • the at least one monocistronic RNA and the at least one bi- or even multicistronic RNA may preferably also encode (in part) identical antigens selected from one of the above mentioned groups or subgroups of antigens, preferably in one of the above mentioned combinations, provided that the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection as a whole provides at least two (preferably different) antigens as defined above.
  • Such an aspect may be advantageous e.g. for a staggered, e.g. time dependent, administration of the inventive solution for lyophilization, transfection and/or injection, e.g.
  • RNAs encoding the at least two (preferably different) antigens may be e.g. contained in (different parts of) a kit of parts composition or may be e.g. administered separately as components of different pharmaceutical compositions, vaccines, lyophilized nucleic acids, etc.
  • one further class of antigens as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection, 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 protein or peptide antigens and their fragments or epitopes.
  • antigens derived from animals which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection 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, cherry compassion 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, aniseed, from crop,
  • Antigens derived from fungi which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may include antigens derived from, without being limited thereto, e.g. Alternia 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection may include antigens derived from, without being limited thereto, e.g. Bacillus tetani, Staphylococcus aureus, Streptomyces griseus, etc. Antibodies
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • the term “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 lyophilized nucleic acid according to the present invention as well.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • each lyophi lized nucleic acid encoding a specific (monoclonal) antibody being directed to specific antigens or immunogens or epitopes of a protein.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed to different determinants (epitopes), 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 (1 975), or may be made by recombinant DNA methods, e.g. as described in U.S. Pat. No. 4,81 6,567.
  • “Monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et a/., Nature, 348:552-554 (1 990), 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 (hybridomas) 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 at least one nucleic acid (sequence) encoding these antibodies can be present as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection.
  • non-human monoclonal or polyclonal antibodies such as murine antibodies may also be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection such antibodies are typically only of limited use, since they generally induce an immune response by production of human antibodies directed to the said non-human antibodies, in the human body. Therefore, a particular non-human antibody can only be administered once to the human.
  • chimeric, humanized non- human and human antibodies are also envisaged encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection.
  • Chimeric antibodies which may be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • Swiss Humanized antibodies which may be also encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 at least one nucleic acid (sequence) 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • bispecificity means herewith that the antigen-binding regions of the antibodies are specific for two different epitopes.
  • different antigens, immunogens or epitopes, etc. can be brought close together, what, optionally, allows a direct interaction of the two components.
  • different cells such as effector cells and target cells can be connected via a bispecific antibody.
  • antibodies or fragments thereof which bind, on the one hand, a soluble antigen as described herein, and, on the other hand, an antigen or receptor on the surface of a tumor cell.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may also encode intrabodies, wherein these intrabodies may be antibodies as defined above. Since these antibodies are intracellular expressed antibodies, i.e. antibodies which may be encoded by nucleic acids localized in specific areas of the cell and also expressed there, such antibodies may be termed intrabodies.
  • Antibodies as encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • a 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.
  • a 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection may be in the form of dsRNA, preferably siRNA.
  • a dsRNA, or a siRNA is of interest particularly in connection with the phenomenon of RNA interference.
  • RNAi RNA interference
  • 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 ).
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may thus be a double-stranded RNA (dsRNA) having a length of from 1 7 to 29, preferably from 1 9 to 25, and preferably being at least 90%, more preferably 95% and especially 100% (of the nucleotides of a dsRNA) complementary to a section of the nucleic acid (sequence) of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore, either a coding or a non-coding section, preferably a coding section.
  • dsRNA double-stranded RNA
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be a dsRNA having the general structure 5'-(N 17 . 29 )-3', preferably having the general structure 5'-(N 19 .
  • each N is a (preferably different) nucleotide of a section of the mRNA of a therapeutically relevant protein or antigen described hereinbefore, preferably being selected from a continuous number of 1 7 to 29 nucleotides of the mRNA of a therapeutically relevant protein or antigen and being present in the general structure 5'-(N 1 7 _ 2 9)-3 ' in their natural order.
  • dsRNAs used as nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection can also be directed against nucleotide sequences of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore that do not lie in the coding region, in particular in the 5' non-coding region of the mRNA, for example, therefore, against non-coding regions of the mRNA having a regulatory function.
  • the target sequence of the dsRNA used as nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection can therefore lie in the translated and untranslated region of the mRNA and/or in the region of the control elements of a protein or antigen described hereinbefore.
  • the target sequence of a dsRNA used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 mRNA.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be in the form of a CpG nucleic acid, in particular CpG-RNA or CpG-DNA.
  • a CpG-RNA or CpG-DNA used according to the invention 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 used according to the invention is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleic acids have a length as described above. Preferably the CpG motifs are unmethylated.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be in the form of an immunostimulatory RNA.
  • an immunostimulatory RNA used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be any (double-stranded or single-stranded) RNA, e.g. a coding RNA, as defined above.
  • the immunostimulatory RNA may be a single-stranded, a double-stranded or a partially double-stranded RNA, more preferably a single-stranded RNA, and/or a circular or linear RNA, more preferably a linear RNA. More preferably, the immunostimulatory RNA may be a (linear) single-stranded RNA. Even more preferably, the immunostimulatory RNA may be a ((linear) single-stranded) messenger RNA (mRNA). An immunostimulatory RNA may also occur as a short RNA oligonucleotide as defined above.
  • An immunostimulatory RNA as used herein may furthermore be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an immune response.
  • an immune response may occur in various ways.
  • a substantial factor for a suitable immune response is the stimulation of different T-cell sub-populations.
  • T- lymphocytes are typically divided into two sub-populations, the T-helper 1 (Thl ) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Th1 ) and extracellular (Th2) pathogens (e.g. antigens).
  • the two Th cell populations differ in the pattern of the effector proteins (cytokines) produced by them.
  • Th1 cells assist the cellular immune response by activation of macrophages and cytotoxic T- cells.
  • Th2 cells promote the humoral immune response by stimulation of the B-cells for conversion into plasma cells and by formation of antibodies (e.g. against antigens).
  • the Th1/Th2 ratio is therefore of great importance in the immune response.
  • the Th1/Th2 ratio of the immune response is preferably shifted in the direction towards the cellular response (Th1 response) and a cellular immune response is thereby induced.
  • the immune system may be activated by ligands of Toll-like receptors (TLRs).
  • TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen- associated molecular patterns (PAMPs) and play a critical role in innate immunity in mammals.
  • PRR pattern recognition receptor
  • TLR1 - TLR13 Toll-like receptors: TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13
  • TLR1 - TLR13 Toll-like receptors: TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13
  • CpG DNA unmethylated bacterial DNA and synthetic analogs thereof
  • ligands for certain TLRs include certain nucleic acid molecules and that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner, wherein these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc.
  • these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc.
  • Lipford eta/ determined certain G,U- containing oligoribonucleotides as immunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280).
  • RNA sources including ribosomal RNA, transfer RNA, messenger RNA, and viral RNA.
  • any RNA molecule as e.g. defined above (irrespective of its specific length, strandedness, modification and/or nucleotide sequence) may have immunostimulatory properties, i.e. enhance the immune response.
  • An RNA as defined above and being the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may thus be used to enhance (unspecific) immunostimulation, if suitable and desired for a specific treatment.
  • the at least one (immunostimulatory) RNA (molecule) used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may thus comprise any RNA sequence known to be immunostimulatory, including, without being limited thereto, RNA sequences representing and/or encoding ligands of TLRs, preferably selected from family members TLR1 - TLR13, more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-I or MAD-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol.
  • immunostimulatory RNA molecules used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may include any other RNA capable of eliciting an immune response.
  • an immunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA).
  • Such further (classes of) immunostimulatory RNA molecules which may be used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, without being limited thereto, may comprise e.g. an RNA molecule 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
  • I is an integer from 1 to 40
  • n is an integer and is at least 3;
  • n is an integer from 1 to 40
  • n > 1 at least 50% of the nucleotides are guanosine or an analogue thereof.
  • 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
  • I is an integer from 1 to 40
  • n is an integer and is at least 3;
  • n is an integer from 1 to 40
  • n > 1 at least 50% of the nucleotides are cytosine or an analogue thereof.
  • the immunostimulatory RNA molecules used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may comprise a length as defined above in general for RNA molecules of the RNA of the present invention, more preferably a length of 5 to 5000, of 500 to 5000 or, more preferably, of 1000 to 5000 or, alternatively, of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or, more preferably, of 5 to 30 nucleotides.
  • the immunostimulatory RNA used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be furthermore modified, preferably "chemically modified” in order to enhance the immunostimulatory properties of said RNA.
  • the term "chemical modification” means that the immuostimulatory RNA is modified by replacement, insertion or removal of individual or several atoms or atomic groups compared with naturally occurring RNA species.
  • the chemical modification of the immunostimulatory RNA comprises at least one analogue of naturally occurring nucleotides.
  • nucleotide analogues examples which may be mentioned for nucleotide analogues and which may be used herein for modification are analogues of guanosine, uracil, adenosine, thymidine, cytosine.
  • the modifications may refer to modifications of the base, the ribose moiety and/or the phosphate backbone moiety.
  • analogues of guanosine, uracil, adenosine, and cytosine 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'-Ara
  • analogue as described above, particular preference is given according to the invention to those analogues that increase the immunogenicity of the immunostimulatory RNA sequence used as the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection and/or do not interfere with a further modification that has been introduced into said immunostimulatory RNA.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection as defined above may also occur in the form of a modified nucleic acid, wherein any modification, as defined herein, may be introduced into the nucleic acid prior to lyophilization, transfection and/or injection. Modifications as defined herein preferably lead to a further stabilized nucleic acid as defined herein.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may thus be provided as a "stabilized nucleic acid", preferably as a stabilized RNA, more preferably as an RNA 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection.
  • 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 at least one nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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.
  • Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-0-(1 - thiophosphate)).
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro- oligoribonucleotide (2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine- 5 '-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'-amino-2'-deoxycytidine-5'- triphosphate, 2'-amino-2 '-deoxyuridine-5'-triphosphate), 2'-0-alkyl oligoribonucleotide,
  • Significant in this case means an increase in the expression of the protein compared with the expression of the native nucleic acid (sequence) 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- iodouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'- triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5 '-triphosphate,
  • 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.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection can likewise be modified (and preferably stabilized) by introducing further modified nucleotides containing modifications of their ribose or base moieties.
  • nucleotide analogues are defined as non-natively occurring variants of naturally occurring nucleotides.
  • 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 backbone (see above) of the nucleic acid sequence.
  • Exemplary analogues of guanosine, uracil, adenosine, and cytosine 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'-Araadeno
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection can contain a lipid modification.
  • a lipid- modified nucleic acid typically comprises a nucleic acid as defined herein.
  • Such a lipid- modified nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection typically further comprises at least one linker covalently linked with that nucleic acid, and at least one lipid covalently linked with the respective linker.
  • the lipid-modified nucleic acid comprises an at least one nucleic acid as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid.
  • the lipid-modified nucleic acid comprises a nucleic acid RNA as defined herein, at least one linker covalently linked with that nucleic acid, 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.
  • the lipid which may be contained in the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection (complexed or covalently bound thereto) 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.
  • vitamin E alpha-tocopherol
  • RRR-alpha-tocopherol originally D-alpha- tocopherol
  • L-alpha-tocopherol the racemate D,L-alpha-tocopherol
  • vitamin E succinate VES
  • 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 within the scope of the present invention include, without implying any limitation, polyalkylene glycols (Oberhauser et al., Nucl.
  • Acids Res., 1 992, 20, 533) 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 al., EMBO J, 1991 , 10, 1 1 1 ; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids such as, for example, phosphatidylglycerol, diacylphosphatidylglycerol, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
  • polyamines or polyalkylene glycols such as, for example, polyethylene glycol (PEG) (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969), hexaethylene glycol (HEG), palmitin or palmityl residues (Mishra et al, Biochim. Biophys. Acta, 1 995, 1264, 229), octadecy I amines or hexylamino-carbonyl-oxycholesterol residues (Crooke et al, J. Pharmacol. Exp. Then, 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 et al,
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may likewise be stabilized in order to prevent degradation of the nucleic acid by various approaches, particularly, when RNA or mRNA is used as a nucleic acid for the inventive purpose.
  • RNAases ribonucleases
  • the natural degradation of mRNA in the cytoplasm of cells is very finely regulated and RNase contaminations may be generally removed by special treatment prior to use of said compositions, in particular with diethyl pyrocarbonate (DEPC).
  • DEPC diethyl pyrocarbonate
  • a number of mechanisms of natural degradation are known in this connection in the prior art, which may be utilized as well.
  • the terminal structure is typically of critical importance for an mRNA.
  • cap structure a modified guanosine nucleotide
  • the so-called poly-A tail is typically a sequence of up to 200 adenosine nucleotides
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection can therefore be stabilized against degradation by RNases by the addition of a so-called "5' cap” structure.
  • m7G(5')ppp 5'(A,G(5')ppp(5')A or G(5')ppp(5')G as the 5' cap" structure.
  • such 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 (sequence) of the inventive solution for lyophilization, transfection and/or injection, if provided as a mRNA or if the modification does not interfere with the immunogenic properties of the (unmodified or chemically modified) nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection.
  • a modification for example a lipid modification
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may contain, especially if the nucleic acid is in the form of a mRNA, 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.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may contain, especially if the nucleic acid is in the form of a mRNA, 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 to 40 cytosine nucleotides.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be modified, and thus stabilized, especially if the nucleic acid is in the form of a mRNA, by modifying the G/C content of the nucleic acid, particularly an mRNA, preferably of the coding region thereof.
  • the G/C content of the coding region of the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is modified, particularly increased, compared to the G/C content of the coding region of its particular wild type mRNA, i.e. the unmodified mRNA.
  • the encoded amino acid sequence of the at least one mRNA is preferably not modified compared to the coded amino acid sequence of the particular wild type mRNA.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of that mRNA.
  • composition and the sequence of various nucleotides are 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 of the mRNA are therefore varied compared to its wild type mRNA, 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 amino acid to be encoded by the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection especially if the nucleic acid is in the form of a mRNA, there are various possibilities for modification of the at least one mRNA sequence, compared to its wild type sequence.
  • codons which contain A and/or U nucleotides can be modified by substitution of other codons which code for the same amino acids but contain no A and/or U. Examples of these are: the codons for Pro can be modified from CCU or CCA to CCC or CCG;
  • the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be modified from GCU or GCA to GCC or GCG;
  • the codons for Gly can be modified from GGU or GGA to GGC or GGG.
  • the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG;
  • the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be modified from UAU to UAC;
  • the codon for Cys can be modified from UGU to UGC;
  • the codon for His can be modified from CAU to CAC;
  • the codon for Gin can be modified from CAA to CAG;
  • the codons for lie can be modified from AUU or AUA to AUC;
  • codons for Thr can be modified from ACU or ACA to ACC or ACG;
  • the codon for Asn can be modified from AAU to AAC;
  • the codon for Lys can be modified from AAA to AAG;
  • the codons for Val can be modified from GUU or GUA to GUC or GUG; the codon for Asp can be modified from GAU to GAC;
  • the codon for Glu can be modified from GAA to GAG;
  • the stop codon UAA can be modified to UAG or UGA.
  • the codons for Met (AUG) and Trp (UGG) on the other hand, there is no possibility of sequence modification.
  • substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA, compared to its particular wild type mRNA (i.e. the original sequence).
  • all codons for Thr occurring in the wild type sequence can be modified to ACC (or ACG).
  • substitution possibilities are used: substitution of all codons coding for Thr in the original sequence (wild type mRNA) to ACC (or ACG) and
  • the G/C content of the coding region of the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA, 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 coded region of the wild type mRNA.
  • nucleic acid sequence
  • inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA, to the maximum (i.e. 100% of the substitutable codons), in particular in the region coding for a protein, compared to the wild type sequence.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells.
  • nucleic acid sequence of the inventive solution for lyophilization, transfection and/or injection
  • the corresponding modified nucleic acid is translated to a significantly poorer degree than in the case where codons coding for relatively "frequent" tRNAs are present.
  • the coding region of the modified nucleic acid is preferably modified compared to the corresponding region of the wild type mRNA such that at least one codon of the wild type 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.
  • sequences of the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is modified such that codons for which frequently occurring tRNAs are available are inserted.
  • all codons of the wild type 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 modified nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection especially if the nucleic acid is in the form of a mRNA, with the "frequent" codons without modifying the amino acid sequence of the protein encoded by the coding region of the nucleic acid.
  • This preferred aspect allows provision of a particularly efficiently translated and stabilized (modified) nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA.
  • a modified nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection as described above (increased G/C content; exchange of tRNAs) can be carried out using the computer program explained in WO 02/098443 - the disclosure content of which is included in its full scope in the present invention.
  • nucleotide sequence of any desired nucleic acid or mRNA can be modified with the aid of the genetic code or the degenerative nature thereof such that a maximum G/C content results, in combination with the use of codons which code for tRNAs occurring as frequently as possible in the cell, and the amino acid sequence coded by the modified nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection preferably not being modified compared to the non-modified sequence.
  • the source code in Visual Basic 6.0 development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3
  • Microsoft Visual Studio Enterprise 6.0 with Servicepack 3 is also described in WO 02/098443.
  • the A/U content in the environment of the ribosome binding site of the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection is increased compared to the A/U content in the environment of the ribosome binding site of its particular wild type mRNA.
  • This modification an increased A/U content around the ribosome binding site increases the efficiency of ribosome binding to the nucleic acid.
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection especially if the nucleic acid is in the form of a mRNA, may be modified with respect to potentially destabilizing sequence elements.
  • the coding region and/or the 5' and/or 3' untranslated region of this nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection may be modified compared to the particular wild type nucleic acid such that is contains no destabilizing sequence elements, the coded amino acid sequence of the modified nucleic acid of the present invention, especially if the nucleic acid is in the form of a mRNA, preferably not being modified compared to its particular wild type nucleic acid.
  • DSE destabilizing sequence elements
  • nucleic acid sequence of the inventive solution for lyophilization, transfection and/or injection
  • nucleic acid is in the form of a mRNA, optionally in the region which encodes for a protein or a peptide as defined herein
  • one or more such modifications compared to the corresponding region of the wild type nucleic acid can therefore be carried out, so that no or substantially no destabilizing sequence elements are contained there.
  • DSE present in the untranslated regions (3'- and/or 5'-UTR) can also be eliminated from the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA, by such modifications.
  • Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3'-UTR sections of numerous unstable RNAs (Caput et al, Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674).
  • AURES AU-rich sequences
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA, is therefore preferably modified compared to the wild type nucleic acid such that the modified nucleic acid contains no such destabilizing sequences.
  • sequence motifs which are recognized by possible endonucleases, e.g.
  • sequence GAACAAG which is contained in the 3 '-UTR segment of the gene which codes for the transferrin receptor (Binder et al., EMBO J. 1 994, 13: 1 969 to 1980).
  • sequence motifs are also preferably removed in the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection, especially if the nucleic acid is in the form of a mRNA.
  • the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection as defined above especially if the nucleic acid is in the form of a mRNA, has, in a modified form, at least one IRES as defined above and/or at least one 5' and/or 3' stabilizing sequence, in a modified form, e.g. to enhance ribosome binding or to allow expression of different encoded proteins located on an at least one (bi- or even multicistronic) RNA of the nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection as defined above.
  • These stabilizing sequences in the 5' and/or 3' untranslated regions have the effect of increasing the half-life of the nucleic acid in the cytosol.
  • These stabilizing sequences can have 100% sequence identity 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 (alpha-)globin gene e.g.
  • stabilizing sequences which can be used in the present invention for a stabilized nucleic acid.
  • 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: 4), which is contained in the 3'UTR of the very stable RNA which codes for (alpha-)globin, type(l)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik et at., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).
  • nucleic acid (sequence) of the inventive solution for lyophilization, transfection and/or injection as defined above, especially if the nucleic acid is in the form of a mRNA, is therefore preferably present as (alpha-)globin UTR (untranslated regions)-stabilized RNA, in particular as (alpha-)globin UTR-stabilized RNA.

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Abstract

La présente invention concerne (l'utilisation d') une solution contenant au moins un(e) (séquence d') acide(s) nucléique(s) et du mannose libre pour lyophilisation, transfection et/ou injection, en particulier d'ARN et d'ARNm. La solution de l'invention présente un effet positif sur la stabilisation de l'/la (séquence d') acide(s) nucléique(s) pendant la lyophilisation et le stockage mais conduit également à une augmentation considérable de l'efficacité de transfection d'un acide nucléique. Elle augmente donc également l'expression in vivo d'une protéine codée par un tel acide nucléique par l'intermédiaire d'un taux de transfection augmenté. La présente invention concerne en outre un procédé de lyophilisation utilisant la solution contenant du mannose, des compositions pharmaceutiques, des vaccins, des kits, des première et deuxième utilisations médicales appliquant une telle solution contenant du mannose et/ou un(e) tel(le) (séquence d') acide(s) nucléique(s) lyophilisé(e) ou remis(e) en suspension avec une telle solution.
PCT/EP2010/006788 2009-12-09 2010-11-08 Solution contenant du mannose pour la lyophilisation, la transfection et/ou l'injection d'acides nucléiques WO2011069586A1 (fr)

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EP10779700.3A EP2510100B1 (fr) 2009-12-09 2010-11-08 Solution contenant mannose pour la lyophilisation, transfection et/ou injection d'acides nucléiques
US13/509,564 US20120258046A1 (en) 2009-12-09 2010-11-08 Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
US14/492,334 US9616084B2 (en) 2009-12-09 2014-09-22 Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
US15/451,675 US20170182081A1 (en) 2009-12-09 2017-03-07 Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids

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PCT/EP2009/008804 WO2011069529A1 (fr) 2009-12-09 2009-12-09 Solution contenant du mannose pour la lyophilisation, la transfection et/ou l'injection d'acides nucléiques

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US14/492,334 Continuation US9616084B2 (en) 2009-12-09 2014-09-22 Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids

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