WO2011069528A1 - Lyophilisation d'acides nucléiques dans des solutions contenant du lactate - Google Patents

Lyophilisation d'acides nucléiques dans des solutions contenant du lactate Download PDF

Info

Publication number
WO2011069528A1
WO2011069528A1 PCT/EP2009/008803 EP2009008803W WO2011069528A1 WO 2011069528 A1 WO2011069528 A1 WO 2011069528A1 EP 2009008803 W EP2009008803 W EP 2009008803W WO 2011069528 A1 WO2011069528 A1 WO 2011069528A1
Authority
WO
WIPO (PCT)
Prior art keywords
mage
nucleic acid
sequence
lactate
lyophilized
Prior art date
Application number
PCT/EP2009/008803
Other languages
English (en)
Inventor
Thorsten Mutzke
Thomas Ketterer
Florian VON DER MüLBE
Original Assignee
Curevac Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curevac Gmbh filed Critical Curevac Gmbh
Priority to PCT/EP2009/008803 priority Critical patent/WO2011069528A1/fr
Priority to PCT/EP2010/006789 priority patent/WO2011069587A1/fr
Publication of WO2011069528A1 publication Critical patent/WO2011069528A1/fr

Links

Classifications

    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the present invention is directed to the lyophilization of nucleic acids in lactate-containing solutions or formulations.
  • the present invention is particularly suitable for enhancing and improving storage and shipping capabilities of nucleic acids for multiple purposes.
  • the present invention is furthermore directed to methods of lyophilization suitable to prepare such inventive lyophilized nucleic acids, to the use of such lyophilized nucleic acids in the preparation of pharmaceutical compositions, to first and second medical indications using such lyophilized nucleic acids and to kits, particularly to kit of parts, comprising such lyophilized nucleic acids.
  • 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. In this context, the final dosage form providing these nucleic acids but also production, transport and storage thereof are of particular interest.
  • nucleic acids e.g., naked DNA
  • nucleic acids introduced into a patient' circulatory system are typically not stable and therefore have little chance of affecting most disease processes (see e.g. Poxon et a/., Pharmaceutical development and Technology, 5(1 ), 1 1 5- 122 (2000)).
  • This obstacle has led to the development of a number of gene delivery systems, e.g. liposomal delivery, gene delivery vectors, etc., which allow increasing metabolic activities.
  • these systems and formulations are typically not suitable for long-term storage or storage above temperatures of -20°C or even -80°C and thus require methods such as lyophilization to increase shelf life.
  • Lyophilization is a worldwide known and recognized method in the art to enhance storage stability of temperature sensitive biomolecules, such as nucleic acids.
  • 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.
  • free, i.e. unbound, water surrounding the nucleic acid and optionally further components escapes from the solution.
  • Subsequent thereto water being bound on a molecular basis by the nucleic acids may be removed in a secondary drying step by adding thermal energy. In both cases the hydration sphere around the nucleic acids is lost.
  • 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 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.
  • 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.
  • carbohydrates such as sugars play a central role as lyoprotectants.
  • 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 molecules, e.g. RNA.
  • Agents that can substitute for non-freezable water, such as trehalose can demonstrate cryoprotective properties for DNA and other molecules during lyophilization of intact bacteria (see e.g. Israeli et al, Cryobiology, 30, 519-523 (1993); or Rudolph et al, Arch. Biochem. Biophys., 245, 134-143 (1986)).
  • cryoprotective agents such as polyols, amino acids, sugars, and lyotropic salts
  • 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.
  • Poxon et al. (2000, supra) found that a decrease in plasmid DNA activity as measured by an in vitro transfection assay can be ameliorated by the use of carbohydrates during lyophilization of the plasmid DNA.
  • a statistically significant loss of transfection efficiency (p ⁇ 0.05) by lyophilization of pRL-CMV plasmid DNA could completely be restored by using mono- and disaccharides during lyophi lization.
  • lyoprotectants glucose (monosaccaride), sucrose and lactose (disaccharides) were used.
  • Poxon et al. (2000, supra) only carried out investigations with lyophilized plasmid DNA using carbohydrate lyoprotectants. No other nucleic acids, such as RNA or PNA, were discussed. Poxon et al.
  • the plasmid product must be of high purity, essentially in its supercoiled form and free of host-cell proteins, chromosomal RNA, RNA, preferably without the use of RNase A, and endotoxins.
  • Quaak et al. (2008, supra) used as excipients sucrose, trehalose, mannitol and polyvinylpyrrolidone (PVP), wherein lyophilization of formulations containing sucrose as a bulking agent in a concentration of (2%) turned out to result in a stable product.
  • PVP polyvinylpyrrolidone
  • RNAs nucleic acids
  • provision of stabilization of RNAs during lyophilization and long-term storage is particularly important.
  • the physico chemical stability of RNAs in solution is extremely low.
  • RNA is typically completely degraded even in the absence of RNases when stored a few days at room temperature. To avoid such degradation and a loss of function, particularly when regarding coding RNAs such as mRNAs the RNA is to be stored at -20°C or even -80°C.
  • nucleic acids, particularly RNAs as an active agent in a pharmaceutical composition or a vaccine
  • Yadava et a/ (see Yadava et a/., AAPS Pharm. Sci. Tech., Vol. 9, No. 2, June 2008, pp. 335- 341 ) discuss the effect of lyophilization and freeze-thawing on the stability of siRNA- liposome complexes.
  • the lipoplexes in Yadava et a/. (2008, supra) when lyophilized in the presence of sugars, such as glucose or sucrose, could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions, they lost 65-75% of their biological functionality. The lyophilization process thus did not appear to alter siRNA's intrinsic biological activity.
  • RNA interference is the post-transcriptional gene silencing due to the cleavage of mRNA, triggered by small double stranded RNA molecules (small interfering RNAs, siRNAs), homologous in sequence to the target mRNA, which are typically less sensitive to loss of biological activity due to breakage and (partial) degradation than mRNAs or RNAs in general, as these siRNAs may even exhibit a biological activity, when their (short) sequence is partial ly degraded.
  • siRNAs small interfering RNAs
  • RNAs as active substances, e.g. (tumor)vaccination using RNAs, gene therapy using RNAs, etc.
  • the object of the present invention is solved by the attached claims.
  • the problem underlying the present invention is solved by a lyophilized nucleic acid, which has been lyophilized from a lactate containing solution.
  • the inventive lyophilized nucleic acid (sequence) is prepared using a method as described herein, particularly a method of lyophilization of a nucleic acid according to the present invention.
  • lyophilization also termed cryodesiccation
  • lyophilization is typically understood as a freeze-drying process, which allows removing water from a frozen sample (containing at least one nucleic acid and a lactate containing solution) via sublimation as described below in further detail.
  • a lactate as defined herein may be any lactate available in the art.
  • a lactate within the context of the present invention is defined as a chemical compound, particularly a salt, derived from free lactic acid (lUPAC systematic name: 2- hydroxypropanoic acid), also known as milk acid, including its optical isomers L-(+)-lactic acid, (5)-lactic acid, D-(-)-lactic acid or ( c)-lactic acid, more preferably its biologically active optical isomer L-(+)-lactic acid, wherein the salt or an anion thereof, preferably may be selected from sodium-lactate, potassium-lactate, or Al 3 + -lactate, NH 4 + -lactate, Fe-lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution” in
  • Lactic acid is a chemical compound that plays a role in several biochemical processes. It was first isolated in ⁇ 780 by a Swedish chemist, Carl Wilhelm Scheele, and is a carboxylic acid with a chemical formula of C 3 H 6 0 3 . It has a hydroxyl group adjacent to the carboxyl group, making it an alpha hydroxy acid (AHA). In solution, it can lose a proton from the acidic group, producing the lactate ion CH 3 CH(OH)COO " . Lactic acid is chiral and has two optical isomers.
  • L-(+)-lactic acid or (5)-lactic acid is D-(-)-lactic acid or (A)-lactic acid, wherein L-(+)-lactic acid is the biologically important isomer.
  • L-lactate is constantly produced in animals from pyruvate via the enzyme lactate dehydrogenase (LDH) in a process of fermentation during normal metabolism and exercise.
  • LDH lactate dehydrogenase
  • lactic acid is typically produced via fermentation using among others bacteria such as Lactobacillus bacteria, etc.
  • the lactate containing solution as defined herein which is used for preparation of the inventive lyophilized nucleic acid (sequence) as defined herein, typically comprises a lactate concentration prior to lyophilization in the range of about 3 mM to about 300 mM, preferably in the range of about 5 mM to about 200 mM, more preferably in the range of about 10 mM to about 150 mM, even more preferably about 15 mM to about 35 mM, and most preferably 20 mM to about 31 mM.
  • the lactate containing solution as defined herein which is used for preparation of the inventive lyophilized nucleic acid (sequence) as defined herein, typically comprises a Ringer's lactate concentration (or a concentration of any of the afore mentioned lactate containing solutions) prior to lyophilization e.g. in the range of about 10% (w/w) to about 100% (w/w), e.g.
  • Ringer's lactate (100 % (w/w)) is typically defined as a solution comprising 131 mM Na + , 5,36 mM K + , 1 ,84 mM Ca 2+ , and 28,3 mM Lactate).
  • the present invention is directed to a lyophilized nucleic acid, which has been lyophilized from a lactate containing solution.
  • a lyophilized nucleic acid 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 lyophilized nucleic acid (sequence) may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA).
  • the lyophilized nucleic acid is a RNA. More preferably, the lyophilized nucleic acid (sequence) 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 lyophilized nucleic acid 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.
  • the lyophilized nucleic acid may be a single-stranded nucleic acid molecule.
  • the lyophilized nucleic acid may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.
  • the lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 above or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other 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 above or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other protein or peptide suitable for a specific (therapeutic) application, wherein the coding
  • therapeutically active proteins may be encoded by the lyophilized nucleic acid (sequence) according to the present invention. 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, CM-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.
  • 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. IL-8, MIP-1 , RANTES, CCR5, CXR4, etc., or cytokine specific receptors, such as TNF- Rl, TNF-RII, CD40, OX40 (CD134), Fas, etc.
  • Therapeutically active proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention may also be selected from any of the proteins given in the following: 0ATL3, 0FC3, 0PA3, 0PD2, 4-1 BBL, 5T4, 6Ckine, 707- AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1 , ABCA4, ABCB1 , ABCB1 1 , ABCB2, ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1 , ABCD3, ABCG5, ABCG8, ABL1 , ABO, ABR ACAA1 , ACACA, ACADL, ACADM, ACADS, ACADVL, ACAT1 , ACCPN, ACE, ACHE, ACHM3, ACHM1 , ACLS, ACPI, ACTA1 , ACTC, ACTN4, ACVRL1 , AD2, ADA, ADAMTS13,
  • Therapeutically active proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention may further be selected from apoptotic factors or apoptosis related proteins including AIF, Apaf e.g. Apaf-1 , Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x L , Bcl-x s , bik, CAD, Calpain, Caspase e.g.
  • 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 lyophilized nucleic acid (sequence) according to the present invention, can also be an adjuvant protein.
  • an adjuvant protein is preferably to be understood as any protein, which is capable to elicit an innate immune response as defined herein.
  • such an innate immune response comprises activation of a pattern recognition receptor, such as e.g. a receptor selected from the 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 huma proteins involved in adjuvant effects may be used as well.
  • Human adjuvant proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, typically comprise any huma protein, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a TLR.
  • human adjuvant proteins encoded by the lyophilized nucleic acid (sequence) according to the present invention 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, O 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, DAF, H, I, P and CD
  • 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 of
  • Pathogenic adjuvant proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, typically comprise any pathogenic (adjuvant) protein, which is capable of eliciting an innate immune response (in a mammal), more preferably selected from pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc., and even more preferably from pathogenic adjuvant proteins selected from the group consisting of, without being limited thereto, bacterial proteins, protozoa proteins (e.g. profilin - like protein of Toxoplasma gondii), viral proteins, or fungal proteins, animal proteins, etc.
  • pathogenic (adjuvant) protein which is capable of eliciting an innate immune response (in a mammal)
  • pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc. and even more preferably from pathogenic adjuvant proteins selected from the group consist
  • bacterial (adjuvant) proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, may comprise any bacterial protein, which is capable of eliciting an innate immune response (preferably in a mammal) or shows an adjuvant character.
  • such bacterial (adjuvant) proteins are selected from the group consisting of bacterial heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90, 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 lyophilized nucleic acid (sequence) according to the present invention, may also be selected from bacterial adjuvant proteins, even more preferably selected from the group consisting of, without being limited thereto, bacterial flagellins, including flagellins from organisms including Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella, Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte, 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, Agrobacterium
  • Bacterial flagellins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, even more preferably comprise a sequence selected from the group comprising any of the following sequences as referred to their accession numbers:
  • Rhizobium Rhizobium me/iloti flaA M24526 Gl:152220 flaB
  • Salmonella Salmonella D13689 NCBI Gl:21 7062 typhimurium ID
  • Protozoa proteins which may also be encoded by the lyophilized nucleic acid (sequence) according to the present invention, may be selected from any protozoa protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoan heat shock proteins, LeIF from Leishmania spp, profi lin— like protein from Toxoplasma gondii, etc.
  • Viral proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, may be selected from any viral protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Respiratory Syncytial Virus fusion glycoprotein (F-protein), envelope protein from MMT virus, mouse leukemia virus protein, Hemagglutinin protein of wild type measles virus, etc.
  • F-protein Respiratory Syncytial Virus fusion glycoprotein
  • envelope protein from MMT virus 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.
  • Fungal proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, may be selected from any fungal protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, fungal immunomodulatory protein (FIP; LZ-8), etc.
  • FIP fungal immunomodulatory protein
  • pathogenic adjuvant proteins which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, 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 lyophilized nucleic acid (sequence) according to the present invention may alternatively encode an antigen.
  • the term "antigen" refers to a substance which is recognized by the immune system and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies as part of an adaptive immune response.
  • the first step of an adaptive immune response is the activation of 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. Each of these cells has a distinct function in eliciting immune responses.
  • 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. These two types of T cells differ in the class of MHC molecule that they recognize.
  • MHC molecule- MHC class I and MHC class II- which differ in their structure and expression pattern on tissues of the body.
  • CD4 + T cells bind to the MHC class 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.
  • MHC class I molecules Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. But not only pathogenic peptides from viruses are presented by MHC class I molecules, also self-antigens like tumour antigens are presented by them. MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. Thereby MHC class I molecules on the surface of cells infected with viruses or other cytosolic pathogens display peptides from these pathogen. The CD8 + T cells that recognize MHC class peptide 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.
  • TH1 cells 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 cells.
  • TH1 cells activate the microbicidal properties of macrophages and induce B cells to make IgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells
  • TH2 cells initiate the humoral response by activating 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention may be antigens generated outside the cell, more typically antigens not derived from the host organism (e.g. a human) itself (i.e. non-self antigens) but rather derived from host cells outside the host organism, e.g.
  • 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 lyophilized nucleic acid (sequence) according to the present invention may be furthermore antigens generated inside the cell, the tissue or the body, e.g. by secretion of proteins, their degradation, metabolism, etc.
  • Such 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.
  • One class of antigens as encoded by the lyophilized nucleic acid (sequence) according to the present invention comprises tumor antigens.
  • Tumor antigens are preferably located on the surface of the (tumor) cell. Tumor antigens may also be selected from proteins, which are overexpressed in tumor cells compared to a normal cell.
  • tumor antigens also includes antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerate but are associated with the supposed tumor.
  • Antigens which are connected with tumor-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g. growth factors, such as VEGF, bFGF etc., are also included herein.
  • 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.
  • tumor antigens 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. They typically result from a tumor specific mutation.
  • TAAs which are more common, are usually presented by both tumor and healthy cells.
  • TAAs which are more common, are usually presented by both tumor and healthy cells.
  • TAAs which are more common, are usually presented by both tumor and healthy cells.
  • TAAs which are more common, are usually presented by both tumor and healthy cells.
  • tumor antigens are recognized and the antigen-presenting cell can be destroyed by cytotoxic T cells.
  • 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
  • tumor antigens as encoded by the lyophilized nucleic acid (sequence) according to the present invention 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.
  • 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 B1 10 cancer, lung cancer
  • DAM-6/MAGE- differentiation antigen melanoma melanoma skin tumors, ovarian B2 6 cancer, lung cancer
  • 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, 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
  • 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
  • tongue cancer hepatocellular carcinomas, melanoma, gastric cancer, esophageal, colon cancer,
  • bladder cancer bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A3 melanoma antigen-A3 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MACE-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,
  • bladder cancer bladder cancer, melanoma, renal
  • bladder cancer bladder cancer, melanoma, sarcoma, brain tumor, esophagel cancer, renal
  • SART-1 1 cancer lung cancer, uterine cancer head and neck cancer, lung cancer, squamous antigen rejecting tumor renal cell carcinoma, melanoma,
  • gastric cancer colon cancer
  • lung cancer breast cancer
  • breast cancer ovarian
  • the tumor antigens as encoded by the lyophilized nucleic acid (sequence) according to the present invention 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, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD
  • the tumor antigens as encoded by the lyophilized nucleic acid (sequence) according to the present invention 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
  • surviving 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_0003708
  • 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_198253
  • 5T4 e.g. 5T4 according to accession number NM_006670
  • NY-Eso-1 e.g. NY-Esol 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 lyophi lized nucleic acid (sequence) according to the present invention may form a cocktail of antigens, e.g. in a vaccine, a pharmaceutical 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 a vaccine, a pharmaceutical 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 lyophilized nucleic acid (sequence) according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three or even four (preferably different) antigens of the following group of antigens:
  • the lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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: a) wherein at least one antigen is selected from:
  • PSA Prostate-Specific Antigen
  • PSMA Prostate-Specific Membrane Antigen
  • PSCA Prostate Stem Cell Antigen
  • the lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention may form a cocktail of antigens, e.g. in a vaccine, a pharmaceutical 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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:
  • the lyophilized nucleic acid (sequence) according to the present invention 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:
  • hTERT, WT1 , 5T4 and NY-ESO-1 or hTERT, WT1 , 5T4 and Survivin, or hTERT, WT1 , 5T4 and MAGE-C2, or hTERT, 5T4, NY-ESO-1 and Survivin, or hTERT, 5T4, NY-ESO-1 and MAGE-C2, or hTERT, NY-ESO-1 , Survivin and MAGE-C2, or WT1 , 5T4, NY-ESO-1 , and Survivin, or WT1 , 5T4, NY-ESO-1 , and MAGE-C2, or WT1 , 5T4, Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or 5T4, NY-ESO-1
  • the tumor antigens as encoded by the lyophilized nucleic acid (sequence) according to the present invention may form a cocktail of antigens, e.g. in in a vaccine, a pharmaceutical 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 lyophilized nucleic acid (sequence) according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens,
  • 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 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:
  • MAGE-A2 and Survivin • MAGE-A2 and Survivin; or MAGE-A2 and MAGE-C1 ; or MAGE-A2 and MAGE-C2; or 5T4 and MAGE-A3; or
  • MAGE-A3 and MUC1 MAGE-A3 and Her-2/neu; or MAGE- A3 and NY-ESO-1 ; or MAGE-A3 and CEA; or MAGE-A3 and Survivin; or MAGE-A3 and MAGE-C1 MAGE- A3 and MAGE-C2 MUC1 and Her-2/neu; or MUC1 and NY-ESO-1 ; or MUC1 and CEA; or
  • MUC1 and Survivin or MUC1 and MAGE-C1 ; or MUC1 and MAGE-C2; or HER-2/NEU and NY-ESO-1 ; or 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
  • CEA and MAGE-C1 or CEA and MAGE-C2; or Survivin and MAGE-C1 ; or Survivin and MAGE-C2; or MAGE-C1 and MAGE-C2; or hTERT, WT1 and MAGE-A2; or hTERT, WT1 and 5T4; or hTERT, WT1 and MAGE- A3; or hTERT, WT1 and MUC1 ; or hTERT, VVT1 and Her-2/neu; or • hTERT, WT1 and NY-ESO-1 ; or
  • VVT1 VVT1 , MAGE-A2 and Her-2/neu
  • VVT1 VVT1 , MAGE-A2 and Survivin
  • WT1 WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and Survivin; or
  • WT1 WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C1 ; or
  • WTl WTl , MAGE-A2, 5T4, MAGE-A3, MUCl , Her-2/neu, NY-ESO-1 , CEA and MAGE-C2; or • MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C1 ; or
  • VVT1 VVT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin 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 lyophilized nucleic acid (sequence) of the present invention 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 lyophi lized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid (sequence) of the present invention as a whole provides at least two (preferably different) antigens as defined above.
  • Such an embodiment may be advantageous e.g. for a staggered, e.g. time dependent, administration of e.g. a vaccine, a pharmaceutical composition or a kit of the present invention to a patient in need thereof.
  • 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 embodiments according to the present invention.
  • one further class of antigens as encoded by the lyophilized nucleic acid (sequence) according to the present invention comprises allergy antigens.
  • allergy antigens may be selected from antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergy antigens typically belong to different classes of compounds, such as nucleic acids and their fragments, proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc.
  • antigens which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, i.e. protein or peptide antigens and their fragments or epitopes, or nucleic acids and their fragments, particularly nucleic acids and their fragments, encoding such protein or peptide antigens and their fragments or epitopes.
  • antigens derived from animals which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, may include antigens derived from, without being limited thereto, insects, such as mite (e.g. house dust mites), mosquito, bee (e.g. honey bee, bumble bee), cockroache, tick, moth (e.g.
  • mite e.g. house dust mites
  • mosquito e.g. honey bee, bumble bee
  • cockroache e.g.
  • Antigens derived from plants 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, such as oat,
  • Antigens derived from fungi which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, 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 lyophilized nucleic acid (sequence) according to the present invention, may include antigens derived from, without being limited thereto, e.g. Bacillus tetani, Staphylococcus aureus, Streptomyces griseus, etc.. Antibodies
  • the lyophilized nucleic acid (sequence) according to the present invention may encode an antibody.
  • an antibody may be selected from any antibody, e.g. any recombinantly produced or naturally occurring antibodies, known in the art, in particular antibodies suitable for therapeutic, diagnostic or scientific purposes, or antibodies which have been identified in relation to specific cancer diseases.
  • 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 (sequence) according to the present invention as well.
  • the lyophilized nucleic acid (sequence) according to the present invention may encode a polyclonal antibody.
  • polyclonal antibody typically means mixtures of antibodies directed to specific antigens or immunogens or epitopes of a protein which were generated by immunization of a host organism, such as a mammal, e.g. including goat, cattle, swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse, hamster and rabbit.
  • 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 lyophilized nucleic acid (sequence) encoding a specific (monoclonal) antibody being directed to specific antigens or immunogens or epitopes of a protein.
  • the lyophilized nucleic acid (sequence) according to the present invention may encode a monoclonal antibody.
  • the term "monoclonal antibody” herein typically refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed to a single antigenic site. 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 (1975), 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 al., Nature, 348:552-554 (1990), for example.
  • an immunogen (antigen) of interest is injected into a host such as a mouse and B-cell lymphocytes produced in response to the immunogen are harvested after a period of time.
  • the B-cells are combined with myeloma cells obtained from mouse and introduced into a medium which permits the B-cells to fuse with the myeloma cells, producing hybridomas. These fused cells (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 nucleic acid sequences encoding these antibodies can be present as the lyophilized nucleic acid (sequence) according to the present invention.
  • non-human monoclonal or polyclonal antibodies such as murine antibodies may also be encoded by the lyophilized nucleic acid (sequence) according to the present invention.
  • lyophilized nucleic acid sequence
  • 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 lyophilized nucleic acid (sequence) according to the present invention.
  • “Chimeric” antibodies which may be encoded by the lyophilized nucleic acid (sequence) according to the present invention, are preferably antibodies in which the constant domains of an antibody described above are replaced by sequences of antibodies from other organisms, preferably human sequences.
  • “SearchHumanized” (non- human) antibodies which may be also encoded by lyophilized nucleic acid (sequence) according to the present invention, are antibodies in which the constant and variable domains (except for the hypervariable domains) described above of an antibody are replaced by human sequences.
  • the lyophilized nucleic acid (sequence) according to the present invention may encode human antibodies, i.e. antibodies having only human sequences.
  • human antibodies can be isolated from human tissues or from immunized non-human host organisms which are transgene for the human IgG gene locus, and nucleic acid sequences may be prepared according to procedures well known in the art. Additionally, human antibodies can be provided by the use of a phage display.
  • the lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention may also encode intrabodies, wherein these intrabodies may be antibodies as defined above. Since these antibodies are intracellular expressed antibodies, i.e. antibodies which 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 lyophilized nucleic acid (sequence) according to the present invention may preferably comprise full-length antibodies, i.e. antibodies composed of the full heavy and full light chains, as described above. However, derivatives of antibodies such as antibody fragments, variants or adducts may also be encoded by the lyophilized nucleic acid (sequence) according to the present invention.
  • the lyophilized nucleic acid (sequence) according to the present invention 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
  • a scFv single chain variable fragment
  • the domains are linked by an artificial linkage, in general a polypeptide linkage such as a peptide composed of 1 5-25 glycine, proline and/or serine residues.
  • the lyophilized nucleic acid (sequence) according to the present invention may be in the form of dsRNA, preferably si RNA.
  • a dsRNA, or a si RNA 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 lyophilized nucleic acid (sequence) according to the present invention 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 1 00% (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 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 1 00% (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
  • 90% complementary means that with a length of a dsRNA described herein of, for example, 20 nucleotides, this contains not more than 2 nucleotides without corresponding complementarity with the corresponding section of the mRNA.
  • the sequence of the double-stranded RNA used according to the invention is, however, preferably wholly complementary in its general structure with a section of the nucleic acid of a therapeutically relevant protein or antigen described hereinbefore.
  • the lyophilized nucleic acid (sequence) according to the present invention may be a dsRNA having the general structure 5'-(N 1 7 .29)-3 ', preferably having the general structure 5'-(N, 9 _2 5 )-3 ', more preferably having the general structure 5'- ( ⁇ , 9 .
  • 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 17 . 29 )-3' in their natural order.
  • all the sections having a length of from 1 7 to 29, preferably from 19 to 25, base pairs that occur in the mRNA can serve as target sequence for a dsRNA herein.
  • dsRNAs used as lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention may be in the form of an immunostimulatory RNA.
  • the immunostimulatory RNA of the complexed RNA of the present invention 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.
  • 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. In this context, 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 (Thi ) 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 et a/ determined certain G,U-containing oligoribonucleotides as immunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280).
  • the immunostimulatory G,U-containing oligoribonucleotides described by Lipford et a/ were believed to be derivable from RNA sources including ribosomal RNA, transfer RNA, messenger RNA, and viral RNA.
  • RNA molecule
  • immunostimulatory properties i.e. enhance the immune response.
  • RNA as defined above and being the lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention, may include any other RNA capable of eliciting an immune response.
  • 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 lyophilized nucleic acid (sequence) according to the present invention, 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 lyophilized nucleic acid (sequence) according to the present invention 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 lyophilized nucleic acid (sequence) according to the present invention may be furthermore modified, preferably "chemically modified” in order to enhance the immunostimulatory properties of said DNA.
  • 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'-Araaden
  • the lyophilized nucleic acid (sequence) according to the present invention 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. Modifications as defined herein preferably lead to a further stabilized lyophilized nucleic acid (sequence) of the present invention.
  • the lyophilized nucleic acid (sequence) according to the present invention as defined above may thus be provided as a "stabi lized nucleic acid", preferably as a stabilized RNA, more preferably as an RNA that is essentially resistant to in vivo degradation (e.g.
  • Such stabilization can be effected, for example, by a modified phosphate backbone of the lyophilized nucleic acid (sequence) according to the present invention.
  • a backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the lyophilized nucleic acid (sequence) 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 lyophilized nucleic acids may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form.
  • non-ionic phosphate analogues such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group
  • phosphodiesters and alkylphosphotriesters in which the charged oxygen residue is present in alkylated form.
  • backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-0-(1 - thiophosphate)).
  • the lyophilized nucleic acid (sequence) of the present invention may additionally or alternatively also contain sugar modifications.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the lyophilized nucleic acid (sequence) and typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2 '-deoxy-2 '-fluoro- oligoribonucleotide (2 '-fluoro-2'-deoxycytidine-5'-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, 2'- deoxy-2 '-C-
  • 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.
  • the lyophilized nucleic acid (sequence) of the present invention 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'-Araaden
  • the lyophilized nucleic acid (sequence) of the present invention can contain a lipid modification.
  • a lipid-modified lyophilized nucleic acid (sequence) typically comprises a nucleic acid as defined herein.
  • Such a lipid- modified lyophilized nucleic acid (sequence) 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 lyophilized nucleic acid (sequence) 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 lyophilized 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 contained in the lyophilized nucleic acid (sequence) of the present invention is typically a lipid or a lipophilic residue that preferably is itself biologically active.
  • Such lipids preferably include natural substances or compounds such as, for example, vitamins, e.g. alpha-tocopherol (vitamin E), including RRR-alpha-tocopherol (formerly D-alpha-tocopherol), L-alpha-tocopherol, the racemate D,L-alpha-tocopherol, vitamin E succinate (VES), or vitamin A and its derivatives, e.g. retinoic acid, retinol, vitamin D and its derivatives, e.g.
  • vitamins e.g. alpha-tocopherol (vitamin E), including RRR-alpha-tocopherol (formerly D-alpha-tocopherol), L-alpha-tocopherol, the racemate D,L-alpha-tocopherol, vitamin E succinate (VES), or
  • 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.
  • aliphatic groups such as, for example, C1 -C20-alkanes, C1 -C20-alkenes or C1 -C20-aikanol 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,
  • phospholipids such as, for
  • 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), octadecylamines or hexylamino- carbonyl-oxycholesterol residues (Crooke et al, J. Pharmacol. Exp.
  • the lyophilized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid. It is known in the art that instability and (fast) degradation of mRNA or of RNA in general may represent a serious problem in the application of RNA based compositions.
  • RNAases RNA-degrading enzymes
  • RNase contaminations may be generally removed by special treatment prior to use of said compositions, in particular with diethyl pyrocarbonate (DEPC).
  • DEPC diethyl pyrocarbonate
  • cap structure a modified guanosine nucleotide
  • poly-A tail a sequence of up to 200 adenosine nucleotides
  • the lyophilized nucleic acid (sequence) of the present invention can therefore be stabilized against degradation by RNases by the addition of a so-called "5' cap” structure.
  • a so-called "5' cap” structure Particular preference is given in this connection to an m7G(5')ppp (5'(A,G(5')ppp(5')A or G(5')ppp(5')G as the 5' cap" structure.
  • a modification is introduced only if a modification, for example a lipid modification, has not already been introduced at the 5' end of the lyophilized nucleic acid (sequence) of the present invention if provided as a mRNA or if the modification does not interfere with the immunogenic properties of the (unmodified or chemically modified) lyophilized nucleic acid (sequence) of the present invention.
  • the lyophilized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid (sequence) of the present invention 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.
  • This modification of the lyophilized nucleic acid (sequence) of the present invention is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of that mRNA.
  • the composition and the sequence of various nucleotides is important.
  • sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content.
  • the codons 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).
  • nucleic acid is in the form of a mRNA
  • amino acids which are encoded by codons which contain exclusively G or C nucleotides no modification of the codon is necessary.
  • the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U is present.
  • 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 AG A 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 Phe can be modified from UUU to UUC
  • 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.
  • substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the lyophilized nucleic acid (sequence) of the present invention, 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).
  • combinations of the above 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 lyophilized nucleic acid (sequence) of the present invention is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coded region of the wild type mRNA.
  • At least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70 %, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a protein or peptide as defined herein or its fragment or variant thereof or the whole sequence of the wild type mRNA sequence are substituted, thereby increasing the GC content of said sequence.
  • a further preferred modification of the lyophilized nucleic acid (sequence) of the present invention is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells.
  • the corresponding modified nucleic acid sequence is translated to a significantly poorer degree than in the case where codons coding for relatively "frequent" tRNAs are present.
  • the modified lyophilized nucleic acid (sequence) of the present invention is in the form of a mRNA
  • the coding region of the modified lyophilized nucleic acid (sequence) 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 lyophilized nucleic acid (sequence) of the present invention 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.
  • Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g.
  • the sequential G/C content which is increased, in particular maximized, in the modified lyophilized nucleic acid (sequence) of the present invention 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 embodiment allows provision of a particularly efficiently translated and stabilized (modified) lyophilized nucleic acid (sequence) of the present invention, especially if the nucleic acid is in the form of a mRNA.
  • 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 lyophilized nucleic acid (sequence) of the present invention 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 lyophilized nucleic acid (sequence) of the present invention 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.
  • the lyophilized nucleic acid (sequence) of the present invention may be modified with respect to potentially destabilizing sequence elements.
  • the coding region and/or the 5' and/or 3' untranslated region of this lyophilized nucleic acid (sequence) 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 lyophilized nucleic acid (sequence) 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
  • DSE present in the untranslated regions (3'- and/or 5'-UTR) can also be eliminated from the lyophilized nucleic acid (sequence) of the present invention, especially if the nucleic acid is in the form of a mRNA, by such modifications.
  • destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3'-UTR sections of numerous unstable RNAs (Caput et a/., Proc. Natl. Acad. Sci. USA 1986, 83: 1 670 to 1674).
  • AURES AU-rich sequences
  • the lyophilized nucleic acid (sequence) of the present invention is therefore preferably modified compared to the wild type nucleic acid such that the modified nucleic acid contains no such destabilizing sequences. This also applies to those 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 lyophilized nucleic acid (sequence) of the present invention, especially if the nucleic acid is in the form of a mRNA.
  • the lyophilized nucleic acid (sequence) of the present invention 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 the at least one (bi- or even multicistronic) RNA of the lyophilized nucleic acid (sequence) of the present invention.
  • 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 lyophilized 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 a/., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).
  • the lyophilized nucleic acid (sequence) of the present invention is therefore preferably present as (alpha-)globin UTR (untranslated regions)-stabilized RNA, in particular as (alpha-)globin UTR-stabilized RNA.
  • nucleic acid (sequence) of the present invention substitutions, additions or eliminations of bases are preferably carried out with the lyophilized nucleic acid (sequence) of the present invention, especially if the nucleic acid is in the form of a mRNA, using a DNA matrix for preparation of the nucleic acid lyophilized nucleic acid (sequence) of the present invention by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation strategy (see e.g. Maniatis et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, NY, 2001 ).
  • a corresponding DNA molecule may be transcribed in vitro.
  • This DNA matrix preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence for the nucleic acid, e.g. mRNA, to be prepared and a termination signal for in vitro transcription.
  • the DNA molecule, which forms the matrix of at least one RNA of interest may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria.
  • Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai et a/., Development 1995, 121 : 2349 to 2360), pGEM ® series, e.g. pGEM ® -1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf. also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, FL, 2001 .
  • Nucleic acid molecules used according to the invention as defined above may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase synthesis, as well as in vitro methods, such as in vitro transcription reactions.
  • the lyophilized nucleic acid (sequence) of the present invention especially if the nucleic acid is in the form of a mRNA, may additionally or alternatively encode a secretory signal peptide.
  • signal peptides are sequences, which typically exhibit a length of about 1 5 to 30 amino acids and are preferably located at the N-terminus of the encoded peptide, without being limited thereto.
  • Signal peptides as defined herein preferably allow the transport of the protein or peptide as encoded by the lyophilized nucleic acid (sequence) of the present invention, especially if the nucleic acid is in the form of a mRNA, into a defined cellular association, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non-classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g.
  • signal sequences of cytokines or immunoglobulines as defined herein signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lampl , Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • signal sequences of MHC class I molecule HLA- A*0201 may be used according to the present invention.
  • any of the above modifications may be applied to the lyophilized nucleic acid (sequence) of the present invention, especially if the nucleic acid is in the form of a mRNA, and further to any lyophilized nucleic acid (sequence) as used in the context of the present invention and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective lyophilized nucleic acid.
  • a person skilled in the art will be able to take his choice accordingly.
  • the lyophilized nucleic acid (sequence) as well as proteins or peptides as encoded by the lyophilized nucleic acid (sequence) of the present invention as defined above, may comprise fragments or variants of those sequences.
  • Such fragments or variants may typically comprise a sequence having a sequence identity with one of the above mentioned nucleic acids, or with one of the proteins or peptides or sequences, if encoded by the nucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, to the entire wild type sequence, either on nucleic acid level or on amino acid level.
  • “Fragments” of proteins or peptides in the context of the present invention may comprise a sequence of a protein or peptide as defined above, which is, with regard to its amino acid sequence (or its encoded nucleic acid sequence), N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid sequence). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence identity with respect to such a fragment as defined above may therefore preferably refer to the entire protein or peptide as defined above or to the entire (coding) nucleic acid sequence of such a protein or peptide.
  • fragments of nucleic acids in the context of the present invention may comprise a sequence of a nucleic acid as defined above, which is, with regard to its nucleic acid sequence 5'-, 3'- and/or intrasequentially truncated compared to the nucleic acid sequence of the original (native) nucleic acid sequence.
  • a sequence identity with respect to such a fragment as defined above may therefore preferably refer to the entire nucleic acid as defined above.
  • Fragments of proteins or peptides in the context of the present invention may furthermore comprise a sequence of a protein or peptide as defined above, which has a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 1 3 or more amino acids, e.g.
  • fragments may be selected from any part of the amino acid sequence.
  • These fragments are typically recognized by T- cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
  • Fragments of proteins or peptides as defined herein may also comprise epitopes of those proteins or peptides.
  • Epitopes also called "antigen determinants" in the context of the present invention are typically fragments located on the outer surface of (native) proteins or peptides as defined herein, preferably having 5 to 1 5 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies or B-cell receptors, i.e. in their native form.
  • Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides.
  • antigenic determinants can be conformational or discontinous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • “Variants” of proteins or peptides as defined above may be encoded by the lyophilized nucleic acid (sequence) of the present invention, wherein nucleic acids of the lyophilized nucleic acid (sequence) of the present invention, encoding the protein or peptide as defined above, are exchanged.
  • a protein or peptide may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants have the same biological function or specific activity compared to the full- length native potein, e.g. its specific antigenic property.
  • the lyophilized nucleic acid (sequence) of the present invention may also encode a protein or peptide as defined above, wherein the encoded amino acid sequence comprises conservative amino acid substitution(s) compared to its physiological sequence.
  • the encoded amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined above.
  • Substitutions in which amino acids which originate from the same class are exchanged for one another are called conservative substitutions.
  • these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g.
  • CD spectra circular dichroism spectra
  • variants of proteins or peptides as defined above which may be encoded by the lyophilized nucleic acid (sequence) of the present invention, may also comprise those sequences, wherein nucleic acids of the lyophilized nucleic acid (sequence) are exchanged according to the degeneration of the genetic code, without leading to an alteration of respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • nucleic acid sequences e.g. nucleic acid sequences as defined herein, or amino acid sequences, preferably their encoded amino acid sequences, e.g. the amino acid sequences of the proteins or peptides as defined above
  • the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position.
  • the percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions.
  • the percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et a/. (1993), PNAS USA, 90:5873-5877 or Altschul et a/. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • the nucleic acid as defined above is typically present in a lactate containing solution prior to lyophilization.
  • the lactate is typically already contained in or added to the nucleic acid as defined above to form such a solution, or vice versa.
  • the nucleic acid and lactate containing solution prior to lyophilization may additionally contain water, preferably water for injection (WFI).
  • WFI water for injection
  • WFI water for injection
  • WFI Water for Injection
  • WFI is water purified by distillation or reverse osmosis.
  • WFI is typically produced by either distillation or 2-stage reverse osmosis.
  • WFI typically does not contain more than 0.25 USP endotoxin units (EU) per ml.
  • Endotoxins are a class of pyrogens that are components of the cell wall of Gram-negative bacteria (the most common type of bacteria in water), preferably in an action limit of 10 cfu/100 ml.
  • the microbial quality may be tested by membrane filtration of a 100 ml sample and plate count agar at an incubation temperature of 30 to 35 degrees Celsius for a 48-hour period.
  • the chemical purity requirements of WFI are typically the same as of PW (purified water).
  • the (residual) water content of the lyophilized nucleic acid (sequence) as defined herein is typically reduced to a content of about 0.5 % (w/w) to about 10 % (w/w), more preferably to a content of about 1 % (w/w) to about 5 % (w/w), even more preferably to a content of about 2 % (w/w) to about 4% (w/w), most preferably to a content of about 3 % (w/w), e.g. 3 % (w/w) + 2 % (w/w), or 3 % (w/w) ⁇ 1 % (w/w).
  • the lyophilized nucleic acid (sequence) as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may contain, additional to the nucleic acid and the lactate, further components or additives, e.g. a cryoprotectant, a lyoprotectant or any further suitable additive, preferably as defined in the following.
  • the lyophilized nucleic acid (sequence) as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain at least one suspending agent, preferably mannit, preferably in a concentration of about 1 to 15% (w/w), more preferably in a concentration of about 3 to 10% (w/w), and even more preferably in a concentration of about 4 to 6% (w/w).
  • suspending agent preferably mannit, preferably in a concentration of about 1 to 15% (w/w), more preferably in a concentration of about 3 to 10% (w/w), and even more preferably in a concentration of about 4 to 6% (w/w).
  • the lyophilized nucleic acid (sequence) as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain at least one component or additive selected, e.g., from proteins, amino acids, alcohols, carbohydrates, mannose, mannit, metals or metal ions, surfactants, polymers or complexing agents, buffers, etc., or a combination thereof.
  • one preferred component or additive may also be selected from the group of amino acids.
  • group may comprise, without being limited thereto, any naturally occurring amino acid, including alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine, more preferably glycine, arginine, and alanine.
  • Cryoprotectants and/or lyoprotectants selected from the group of amino acids may additionally comprise any modification of a naturally occurring amino acid as defined above.
  • a further component or additive may be selected from the group of alcohols.
  • Such group may comprise, without being limited thereto, any alcohol suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, mannitol, polyethyleneglycol, polypropyleneglycol, sorbitol, etc.
  • a further component or additive may be selected from the group of carbohydrates.
  • Such group may comprise, without being limited thereto, any carbohydrate, suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, monosaccharides, such as e.g. glucose, fructose, (free) mannose (i.e.
  • disaccharides such as e.g. lactose, maltose, sucrose, trehalose, etc.
  • polysaccharides such as e.g. dextran, HP- beta CD, etc.
  • mannose as a further component or additive 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.
  • the occurrence of the different mannose isomers in nature significantly differs.
  • 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.
  • mannose upon solubilization in an aqueous solution mannose typically forms the above anomers in an equilibrity reaction, typically in the above concentrations.
  • mannose as a further component or additive 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 a further component or additive may be selected from L-mannose or a racemic mixture of D-Mannose and/or L-Mannose, wherein D-mannose is preferably as described above.
  • Such mixtures may be obtained e.g. by a non-selective synthesis of mannose, e.g. by non-selective 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 a further component or additive 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.
  • a further suitable component or additive may be selected from the group of proteins.
  • Such group may comprise, without being limited thereto, proteins such as albumin, gelatine, therapeutically active proteins as defined above, antibodies as defined above, antigens as defined above, or any further protein encoded by the lyophilized nucleic acid (sequence) of the present invention as defined above.
  • a component or additive which may be contained in the lyophilized nucleic acid (sequence) as defined herein and accordingly in the nucleic acid and lactate containing solution prior to lyophilization may be selected from the group of metals or metal ions, typically comprising, without being limited thereto, metals or metal ions or salts selected from
  • alkali metals including members of group 1 of the periodic table: lithium (Li), sodium (Na), potassium (K), rubidium (Rb), caesium (Cs), and francium (Fr), and their (monovalent) metal alkali metal ions and salts; preferably lithium (Li), sodium (Na), potassium (K), and their (monovalent) metal alkali metal ions and salts;
  • alkaline earth metals including members of group 2 of the periodic table: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra), and their (divalent) alkaline earth metal ions and salts; preferably magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and their (divalent) alkaline earth metal ions and salts;
  • transition metals including members of groups 3 to 13 of the periodic table and their metal ions and salts;.
  • the transition metals typically comprise the 40 chemical elements 21 to 30, 39 to 48, 71 to 80, and 103 to 1 12.
  • the name transition originates from their position in the periodic table of elements. In each of the four periods in which they occur, these elements represent the successive addition of electrons to the d atomic orbitals of the atoms. In this way, the transition metals represent the transition between subgroup 2 elements and subgroup 12 (or 13) elements.
  • Transition metals in the context of the present invention particularly comprise members of subgroup 3 of the periodic table: including Scandium (Sc), Yttrium (Y), and Lutetium (Lu), members of subgroup 4 of the periodic table: including Titan (Ti), Zirconium (Zr), and Hafnium (Hf), members of subgroup 5 of the periodic table: including Vanadium (V), Niobium (Nb), and Tantalum (Ta), members of subgroup 6 of the periodic table: including Chrome (Cr), Molybdenum (Mo), and Tungsten (W), members of subgroup 7 of the periodic table: including Manganese (Mn), Technetium (Tc), and Rhenium (Re), members of subgroup 8 of the periodic table: including Iron (Fe), Ruthenium (Ru), and Osmium (Os), members of subgroup 9 of the periodic table: including Cobalt (Co), Rhodium (Rh), and Iridium (Ir), members of subgroup 10 of the periodic table: including Nickel (Ni
  • earth metals or members of the boron group including members of group 3 of the periodic table: including Boron (B), Aluminium (Al), Gallium (Ga), Indium (In) and Thallium (TI) and their metal ions and salts; preferably Boron (B) and Aluminium (Al) and their metal ions and salts;
  • metalloids or semi metals including Boron (B), Silicon (Si), Germanium (Ge), Arsenic (As), Antimony (Sb), Tellurium (Te).and Polonium (Po), and their semi metal ions and salts; preferably Boron (B) and Silicon (Si) and their semi metal ions and salts;
  • a further component or additive may be selected from the group of surfactant may comprise, without being limited thereto, any surfactant, suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, Tween, e.g. Tween 80 (0.2%), Pluronics, e.g. Pluronic L121 (1 .25%), Triton-X, SDS, PEG, LTAB, Saponin, Cholate, etc.
  • Another component or additive which may be contained in the lyophilized nucleic acid (sequence) as defined herein and accordingly in the nucleic acid and lactate containing solution prior to lyophilization may be selected from the group of polymers or complexing agents, typically comprising, without being limited thereto, any polymer suitable for the preparation of a pharmaceutical composition, such as minor/major groove binders, nucleic acid binding proteins, lipoplexes, nanoplexes, non-cationic or non-polycationic compounds, such as PLGA, Polyacetate, Polyacrylate, PVA, Dextran, hydroxymethylcellulose, starch, MMP, PVP, heparin, pectin, hyaluronic acid, and derivatives thereof, or cationic or polycationic compound, particularly cationic or polycationic polymers or cationic or polycationic lipids, preferably a cationic or polycationic polymers.
  • any polymer suitable for the preparation of a pharmaceutical composition such as minor/major groove bind
  • such a cationic or polycationic compound is typically selected from any cationic or polycationic compound, suitable for complexing and thereby stabilizing a nucleic acid as defined herein, e.g. by associating the nucleic acid as defined herein with the cationic or polycationic compound.
  • cationic or polycationic compounds are selected from cationic or polycationic peptides or proteins, including protamine, nucleoline, spermin or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophil), PLL
  • preferred cationic or polycationic proteins or peptides may be selected from following proteins or peptides having the following total formula: (Arg)
  • oligoargi nines in this context are e.g. Arg 7 , Arg 8 , Arg 9 , Arg 7 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • Further preferred cationic or polycationic compounds, which can be used for complexing the nucleic acid as defined herein may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • PEI polyethyleneimine
  • DOTMA [1 -(2,3-sioleyloxy)propyl)]-N,N,N- trimethylammonium chloride, DMRIE, di-Cl 4-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3- (trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(a- trimethylammonioacetyl)diethanolamine chloride, CLIP!
  • modified polyaminoacids such as ⁇ -aminoacid- polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl- 4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanoI polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc.
  • polyallylamine sugar backbone based poly
  • association or complexing the nucleic acid as defined herein with cationic or polycationic compounds preferably provides adjuvant properties to the RNA and confers a stabilizing effect to the nucleic acid as defined herein by complexation.
  • the procedure for stabilizing the nucleic acid as defined herein is in general described in EP-A-1083232, the disclosure of which is incorporated by reference into the present invention in its entirety.
  • cationic or polycationic compounds are compounds selected from the group consisting of protamine, nucleoline, spermin, spermidine, oligoarginines as defined above, such as Arg 7 , Arg 8 , Arg 9 , Arg 7 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • the lyophilized nucleic acid (sequence) as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain water, water for injection (WFI), or a buffer, preferably selected from a buffer as defined above, e.g.
  • a buffer containing 2-hydroxypropanoic acid preferably including at least one of its optical isomers L-(+)-lactic acid, ( )-lactic acid, D-(-)-lactic acid or ( ⁇ -lactic acid, more preferably its biologically active optical isomer L-(+)-lactic acid, or a salt or an anion thereof, preferably selected from sodium-lactate, potassium-lactate, or Al 3 + - lactate, NH 4 + -lactate, Fe-lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or a buffer selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution” in the UK), acetated Ringer ' s solution, or ortho-lactate-containing solutions (e.g.
  • a buffer as defined herein may also be a mannose containing buffer, an isotonic buffer or solution, preferably selected from isotonic saline, a lactate or ortho-lactate-containing isotonic solution, a isotonic buffer or solution selected from phosphate-buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Grey's balanced salt solution (GBSS), or normal saline (NaCI), hypotonic (saline) solutions with addition of glucose or dextrose, or any solution as defined herein, etc.
  • PBS phosphate-buffered saline
  • TRIS-buffered saline TRIS-buffered saline
  • HBSS Hank's balanced salt solution
  • EBSS Earle's balanced salt
  • isotonic buffers or solutions are preferably prepared by a skilled person preferably as defined herein or according to definitions preparation protocols well known in the art for these specific isotonic buffers or solutions. More preferably, the lactate containing solution prior to lyophilization as defined above may contain these isotonic buffers or solutions or (all) its contents in isotonic concentrations, preferably as defined herein or in the art for these specific isotonic solutions.
  • the lactate containing solution prior to lyophilization as defined above and accordingly its components, additives, isotonic buffers or solutions, may be present in an osmolality comparable to that of blood plasma, preferably in an osmolality as generally defined herein for the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid).
  • a buffer may be used, more preferably an aqueous (isotonic solution or aqueous) buffer, containing a sodium salt, preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3 mM of a potassium sait.
  • the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
  • examples of sodium salts include e.g.
  • examples of the optional potassium salts include e.g. KG, Kl, KBr, K 2 C0 3 , KHC0 3 , K 2 S0 4
  • examples of calcium salts include e.g. CaCI 2 , Cal 2 , CaBr 2 , CaC0 3 , CaS0 4 , Ca(OH) 2 .
  • the salts are present in such an (isotonic solution or) buffer in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI) and at least 0.01 mM calcium chloride (CaCl 2 ).
  • organic anions of the aforementioned cations may be contained in the buffer.
  • the buffer may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCI 2 ) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides. CaCl 2 can also be replaced by another salt like KCI.
  • the buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
  • Reference media are e.g.
  • liquids occurring in "in vivd' methods such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids.
  • common buffers or liquids are known to a skilled person.
  • the inventive lyophilized nucleic acid (sequence) when lyophilized, may again be reconstituted after lyophilization in a buffer as defined herein, preferably as defined above, e.g. as a further step of a method for lyophilization as defined herein.
  • the inventive lyophilized nucleic acid when lyophilized, may alternatively be reconstituted in water, a buffer as defined above, or a buffer containing mannose, to obtain the desired salt concentration or alternatively the desired buffer conditions.
  • the reconstitution of the lyophilized nucleic acid is carried out in WFI (water for injection), if the nucleic acid has been lyophilized in Ringer Lactate solution which represents an isotonic solution for injection.

Abstract

La présente invention concerne la lyophilisation d'acides nucléiques dans des solutions ou formulations contenant du lactate. La présente invention est particulièrement adaptée pour renforcer et améliorer les capacités de stockage et de transport d'acides nucléiques pour de multiples applications. La présente invention concerne, en outre, des procédés de lyophilisation adaptés pour préparer de ces acides nucléiques lyophilisés de l'invention, l'utilisation de tels acides nucléiques lyophilisés dans la préparation de compositions pharmaceutiques, des première et deuxième indications médicales utilisant ces acides nucléiques lyophilisés et des kits, en particulier un kit de composants, comprenant ces acides nucléiques lyophilisés.
PCT/EP2009/008803 2009-12-09 2009-12-09 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate WO2011069528A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/EP2009/008803 WO2011069528A1 (fr) 2009-12-09 2009-12-09 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate
PCT/EP2010/006789 WO2011069587A1 (fr) 2009-12-09 2010-11-08 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2009/008803 WO2011069528A1 (fr) 2009-12-09 2009-12-09 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate

Publications (1)

Publication Number Publication Date
WO2011069528A1 true WO2011069528A1 (fr) 2011-06-16

Family

ID=42060970

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2009/008803 WO2011069528A1 (fr) 2009-12-09 2009-12-09 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate
PCT/EP2010/006789 WO2011069587A1 (fr) 2009-12-09 2010-11-08 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/EP2010/006789 WO2011069587A1 (fr) 2009-12-09 2010-11-08 Lyophilisation d'acides nucléiques dans des solutions contenant du lactate

Country Status (1)

Country Link
WO (2) WO2011069528A1 (fr)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102323230A (zh) * 2011-08-16 2012-01-18 南京欣迪生物药业工程有限责任公司 一种精浆果糖浓度检测试剂盒及应用
CN103601665A (zh) * 2013-11-18 2014-02-26 大连杰信生物科技有限公司 一种离子液体双水相体系萃取氨基酸方法
US8664194B2 (en) 2011-12-16 2014-03-04 Moderna Therapeutics, Inc. Method for producing a protein of interest in a primate
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
CN103936979A (zh) * 2013-01-21 2014-07-23 国家纳米科学中心 一种聚(谷氨酸-co-乳酸)-磷脂酰乙醇胺接枝聚合物及其制备方法和应用
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
CN104792978A (zh) * 2014-01-17 2015-07-22 中国科学院生态环境研究中心 一种用于dna氧化损伤产物8-羟基脱氧鸟苷的信号标记分子及标记方法
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9238064B2 (en) 2008-03-03 2016-01-19 University Of Miami Allogeneic cancer cell-based immunotherapy
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
WO2016184575A1 (fr) * 2015-05-20 2016-11-24 Curevac Ag Composition de poudre sèche comprenant de l'arn à chaîne longue
WO2016184576A3 (fr) * 2015-05-20 2016-12-29 Curevac Ag Composition de poudre sèche comprenant de l'arn à chaîne longue
WO2016184577A3 (fr) * 2015-05-20 2017-01-12 Curevac Ag Composition de poudre sèche comprenant de l'arn à chaîne longu
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US9738923B2 (en) 2014-06-18 2017-08-22 Luminex Corporation Methods for generating stabilized lyophilized materials
DE102016107811A1 (de) 2016-04-27 2017-11-02 AstenJohnson PGmbH Industrielles Gewebe, insbesondere Transportband
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US10507183B2 (en) 2011-06-08 2019-12-17 Translate Bio, Inc. Cleavable lipids
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
EP4035659A1 (fr) 2016-11-29 2022-08-03 PureTech LYT, Inc. Exosomes destinés à l'administration d'agents thérapeutiques
US11446250B2 (en) 2015-04-17 2022-09-20 Curevac Real Estate Gmbh Lyophilization of RNA
US11661634B2 (en) 2015-05-08 2023-05-30 CureVac Manufacturing GmbH Method for producing RNA
US11667910B2 (en) 2015-05-29 2023-06-06 CureVac Manufacturing GmbH Method for producing and purifying RNA, comprising at least one step of tangential flow filtration
US11739125B2 (en) 2013-08-21 2023-08-29 Cure Vac SE Respiratory syncytial virus (RSV) vaccine
US11761009B2 (en) 2014-12-12 2023-09-19 CureVac SE Artificial nucleic acid molecules for improved protein expression
WO2023174991A1 (fr) 2022-03-15 2023-09-21 Etherna Immunotherapies Nv Lyophilisation par rotation continue de compositions contenant des acides nucléiques
US11786590B2 (en) 2015-11-09 2023-10-17 CureVac SE Rotavirus vaccines
WO2023218019A1 (fr) 2022-05-12 2023-11-16 Etherna Immunotherapies Nv Composition d'arn lyophilisée
US11833224B1 (en) 2023-02-08 2023-12-05 Leuvian Llc Lyoprotectant compositions and uses thereof
US11865084B2 (en) 2016-12-23 2024-01-09 CureVac SE MERS coronavirus vaccine
US11965000B2 (en) 2023-09-07 2024-04-23 CureVac SE Respiratory syncytial virus (RSV) vaccine

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104672104B (zh) * 2015-01-05 2017-07-21 中山大学 一种天然产物spermatinamine的衍生物及其制备与应用
CN111892643B (zh) * 2020-07-11 2022-07-08 温州大学 双甘肽类铜离子凝胶荧光探针化合物及其制备方法和应用
CN112480229B (zh) * 2020-11-26 2021-07-30 广东省农业科学院植物保护研究所 蛋白Secp43或其编码基因在调控黑腹果蝇雄性生育中的应用
CN114191379A (zh) * 2021-12-17 2022-03-18 西安交通大学医学院第一附属医院 以Pluronic F127为载体进行在体局部转基因的修复皮肤损伤药物及制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020172717A1 (en) * 2000-10-06 2002-11-21 Leong Kam W. Systemic delivery of compounds through non-invasive bladder administration
EP1724353A2 (fr) * 1998-05-12 2006-11-22 The Regents of the University of California Méthode pour la formation de microparticules lipidiques couplées à des protéines
EP1870110A1 (fr) * 2005-03-24 2007-12-26 Dainippon Sumitomo Pharma Co., Ltd. Préparation comprenant des microparticules d'un complexe composé d'une molécule d'acide nucléique et de collagène
US20090220957A1 (en) * 2005-10-31 2009-09-03 Axis-Shield Asa Membrane assay method

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4415732A (en) 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4401796A (en) 1981-04-30 1983-08-30 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
ES2238799T3 (es) 1999-09-09 2005-09-01 Curevac Gmbh Transferencia de arn-m.
EP2842964A1 (fr) 2001-06-05 2015-03-04 Curevac GmbH Méthode virtuelle de détermination d'une séquence d'ARNm modifiée
ES2734652T3 (es) 2002-04-04 2019-12-11 Zoetis Belgium S A Oligorribonucleótidos inmunoestimulantes que contienen G y U

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1724353A2 (fr) * 1998-05-12 2006-11-22 The Regents of the University of California Méthode pour la formation de microparticules lipidiques couplées à des protéines
US20020172717A1 (en) * 2000-10-06 2002-11-21 Leong Kam W. Systemic delivery of compounds through non-invasive bladder administration
EP1870110A1 (fr) * 2005-03-24 2007-12-26 Dainippon Sumitomo Pharma Co., Ltd. Préparation comprenant des microparticules d'un complexe composé d'une molécule d'acide nucléique et de collagène
US20090220957A1 (en) * 2005-10-31 2009-09-03 Axis-Shield Asa Membrane assay method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MAITANI Y ET AL: "Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency", INTERNATIONAL JOURNAL OF PHARMACEUTICS, ELSEVIER BV, NL LNKD- DOI:10.1016/J.IJPHARM.2007.12.033, vol. 356, no. 1-2, 22 May 2008 (2008-05-22), pages 69 - 75, XP022625172, ISSN: 0378-5173, [retrieved on 20071231] *
MOLINA MARION D C ET AL: "The stability of lyophilized lipid/DNA complexes during prolonged storage", JOURNAL OF PHARMACEUTICAL SCIENCES, AMERICAN PHARMACEUTICAL ASSOCIATION, WASHINGTON, US LNKD- DOI:10.1002/JPS.20138, vol. 93, no. 9, 1 September 2004 (2004-09-01), pages 2259 - 2273, XP002527009, ISSN: 0022-3549 *
RIZKALLA N ET AL: "Effect of various formulation parameters on the properties of polymeric nanoparticles prepared by multiple emulsion method", JOURNAL OF MICROENCAPSULATION 200602 GB LNKD- DOI:10.1080/02652040500286185, vol. 23, no. 1, February 2006 (2006-02-01), pages 39 - 57, XP007912678 *
YADAVA PREETI ET AL: "Effect of lyophilization and freeze-thawing on the stability of siRNA-liposome complexes", AAPS PHARMSCITECH, SPRINGER NEW YORK LLC, US LNKD- DOI:10.1208/S12249-007-9000-1, vol. 9, no. 2, 1 June 2008 (2008-06-01), pages 335 - 341, XP002511465, ISSN: 1530-9932, [retrieved on 20071229] *

Cited By (91)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9238064B2 (en) 2008-03-03 2016-01-19 University Of Miami Allogeneic cancer cell-based immunotherapy
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US10064959B2 (en) 2010-10-01 2018-09-04 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9657295B2 (en) 2010-10-01 2017-05-23 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9950068B2 (en) 2011-03-31 2018-04-24 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US10507183B2 (en) 2011-06-08 2019-12-17 Translate Bio, Inc. Cleavable lipids
US10702478B2 (en) 2011-06-08 2020-07-07 Translate Bio, Inc. Cleavable lipids
US11234936B2 (en) 2011-06-08 2022-02-01 Translate Bio, Inc. Cleavable lipids
CN102323230A (zh) * 2011-08-16 2012-01-18 南京欣迪生物药业工程有限责任公司 一种精浆果糖浓度检测试剂盒及应用
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10751386B2 (en) 2011-09-12 2020-08-25 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US8664194B2 (en) 2011-12-16 2014-03-04 Moderna Therapeutics, Inc. Method for producing a protein of interest in a primate
US8680069B2 (en) 2011-12-16 2014-03-25 Moderna Therapeutics, Inc. Modified polynucleotides for the production of G-CSF
US8754062B2 (en) 2011-12-16 2014-06-17 Moderna Therapeutics, Inc. DLIN-KC2-DMA lipid nanoparticle delivery of modified polynucleotides
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9587003B2 (en) 2012-04-02 2017-03-07 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US9050297B2 (en) 2012-04-02 2015-06-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US9254311B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins
US9255129B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9220755B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9221891B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. In vivo production of proteins
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9301993B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides encoding apoptosis inducing factor 1
US9220792B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides encoding aquaporin-5
US9216205B2 (en) 2012-04-02 2015-12-22 Moderna Therapeutics, Inc. Modified polynucleotides encoding granulysin
US9192651B2 (en) 2012-04-02 2015-11-24 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
US9149506B2 (en) 2012-04-02 2015-10-06 Moderna Therapeutics, Inc. Modified polynucleotides encoding septin-4
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9233141B2 (en) 2012-04-02 2016-01-12 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9114113B2 (en) 2012-04-02 2015-08-25 Moderna Therapeutics, Inc. Modified polynucleotides encoding citeD4
US9061059B2 (en) 2012-04-02 2015-06-23 Moderna Therapeutics, Inc. Modified polynucleotides for treating protein deficiency
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9089604B2 (en) 2012-04-02 2015-07-28 Moderna Therapeutics, Inc. Modified polynucleotides for treating galactosylceramidase protein deficiency
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9782462B2 (en) 2012-04-02 2017-10-10 Modernatx, Inc. Modified polynucleotides for the production of proteins associated with human disease
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
CN103936979A (zh) * 2013-01-21 2014-07-23 国家纳米科学中心 一种聚(谷氨酸-co-乳酸)-磷脂酰乙醇胺接枝聚合物及其制备方法和应用
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US11739125B2 (en) 2013-08-21 2023-08-29 Cure Vac SE Respiratory syncytial virus (RSV) vaccine
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
CN103601665A (zh) * 2013-11-18 2014-02-26 大连杰信生物科技有限公司 一种离子液体双水相体系萃取氨基酸方法
CN104792978A (zh) * 2014-01-17 2015-07-22 中国科学院生态环境研究中心 一种用于dna氧化损伤产物8-羟基脱氧鸟苷的信号标记分子及标记方法
US9738923B2 (en) 2014-06-18 2017-08-22 Luminex Corporation Methods for generating stabilized lyophilized materials
US10144954B2 (en) 2014-06-18 2018-12-04 Luminex Corporation Methods for generating stabilized lyophilized materials
US11098344B2 (en) 2014-06-18 2021-08-24 Luminex Corporation Methods for generating stabilized lyophilized materials
US11761009B2 (en) 2014-12-12 2023-09-19 CureVac SE Artificial nucleic acid molecules for improved protein expression
US11491112B2 (en) 2015-04-17 2022-11-08 CureVac Manufacturing GmbH Lyophilization of RNA
US11446250B2 (en) 2015-04-17 2022-09-20 Curevac Real Estate Gmbh Lyophilization of RNA
US11661634B2 (en) 2015-05-08 2023-05-30 CureVac Manufacturing GmbH Method for producing RNA
WO2016184575A1 (fr) * 2015-05-20 2016-11-24 Curevac Ag Composition de poudre sèche comprenant de l'arn à chaîne longue
US11534405B2 (en) 2015-05-20 2022-12-27 Curevac Ag Dry powder composition comprising long-chain RNA
EP3298142B1 (fr) 2015-05-20 2021-07-14 CureVac AG Composition de poudre sèche comprenant de l'arn à chaîne longue
US11179337B2 (en) 2015-05-20 2021-11-23 Curevac Ag Dry powder composition comprising long-chain RNA
US10729654B2 (en) 2015-05-20 2020-08-04 Curevac Ag Dry powder composition comprising long-chain RNA
EP3928800A3 (fr) * 2015-05-20 2022-03-23 CureVac AG Composition de poudre sèche comprenant de l'arn à longue chaîne
EP3916091A3 (fr) * 2015-05-20 2022-03-30 CureVac AG Composition de poudre sèche comprenant de l'arn à longue chaîne
CN107635587A (zh) * 2015-05-20 2018-01-26 库瑞瓦格股份公司 包含长链rna的干燥粉末组合物
US11433027B2 (en) 2015-05-20 2022-09-06 Curevac Ag Dry powder composition comprising long-chain RNA
US10517827B2 (en) 2015-05-20 2019-12-31 Curevac Ag Dry powder composition comprising long-chain RNA
WO2016184577A3 (fr) * 2015-05-20 2017-01-12 Curevac Ag Composition de poudre sèche comprenant de l'arn à chaîne longu
EP3297682B1 (fr) 2015-05-20 2021-07-14 CureVac AG Composition de poudre sèche comprenant de l'arn à chaîne longue
WO2016184576A3 (fr) * 2015-05-20 2016-12-29 Curevac Ag Composition de poudre sèche comprenant de l'arn à chaîne longue
US11667910B2 (en) 2015-05-29 2023-06-06 CureVac Manufacturing GmbH Method for producing and purifying RNA, comprising at least one step of tangential flow filtration
US11760992B2 (en) 2015-05-29 2023-09-19 CureVac Manufacturing GmbH Method for producing and purifying RNA, comprising at least one step of tangential flow filtration
US11834651B2 (en) 2015-05-29 2023-12-05 CureVac Manufacturing GmbH Method for producing and purifying RNA, comprising at least one step of tangential flow filtration
US11786590B2 (en) 2015-11-09 2023-10-17 CureVac SE Rotavirus vaccines
DE102016107811A1 (de) 2016-04-27 2017-11-02 AstenJohnson PGmbH Industrielles Gewebe, insbesondere Transportband
EP4035659A1 (fr) 2016-11-29 2022-08-03 PureTech LYT, Inc. Exosomes destinés à l'administration d'agents thérapeutiques
US11865084B2 (en) 2016-12-23 2024-01-09 CureVac SE MERS coronavirus vaccine
WO2023174991A1 (fr) 2022-03-15 2023-09-21 Etherna Immunotherapies Nv Lyophilisation par rotation continue de compositions contenant des acides nucléiques
WO2023218019A1 (fr) 2022-05-12 2023-11-16 Etherna Immunotherapies Nv Composition d'arn lyophilisée
US11833224B1 (en) 2023-02-08 2023-12-05 Leuvian Llc Lyoprotectant compositions and uses thereof
US11965000B2 (en) 2023-09-07 2024-04-23 CureVac SE Respiratory syncytial virus (RSV) vaccine

Also Published As

Publication number Publication date
WO2011069587A1 (fr) 2011-06-16

Similar Documents

Publication Publication Date Title
US20210060175A1 (en) Methods of immunostimulation with complexes of rna and cationic carriers
US9616084B2 (en) Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
WO2011069528A1 (fr) Lyophilisation d'acides nucléiques dans des solutions contenant du lactate
US20110053829A1 (en) Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
WO2010088927A1 (fr) Utilisation de pei pour l'amélioration de la libération endosomale et de l'expression d'acides nucléiques transfectés, complexés par des composés cationiques ou polycationiques
EP2510100B1 (fr) Solution contenant mannose pour la lyophilisation, transfection et/ou injection d'acides nucléiques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09771306

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09771306

Country of ref document: EP

Kind code of ref document: A1