NUCLEIC ACIDS COMPRISING FORMULA (NuGlXmGnNv)a AND DERIVATIVES THEREOF AS IMMUNOSTIMULATING AGENT/ADJUVANT

Abstract

The present invention relates to nucleic acids of the general formula (I): (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a and derivatives thereof as an immunostimulating agent/adjuvant and to compositions containing same, optionally comprising an additional adjuvant. The present invention furthermore relates to a pharmaceutical composition or to a vaccine, each containing nucleic acids of formula (I) above and/or derivatives thereof as an immunostimulating agent, and optionally at least one additional pharmaceutically active component, e.g. an antigenic agent. The present invention relates likewise to the use of the pharmaceutical composition or of the vaccine for the treatment of cancer diseases, infectious diseases, allergies and autoimmune diseases etc. Likewise, the present invention includes the use of nucleic acids of the general formula (I): (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a and/or derivatives thereof for the preparation of a pharmaceutical composition for the treatment of such diseases.

Claims

1. Nucleic acid molecule of formula (I):
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a, wherein: G is guanosine, uridine or an analogue of guanosine or uridine; X is guanosine, uridine, adenosine, thymidine, cytidine, or an analogue of these nucleotides; N is a nucleic acid sequence having a length of about 4 to 50, each N independently selected from from guanosine, uridine, adenosine, thymidine, cytidine or an analogue of these nucleotides; a is an integer from 1 to 20; l is an integer from 1 to 40, wherein when l=1, G is guanosine or an analogue thereof, when l>1, at least 50% of these nucleotides are guanosine or an analogue thereof; m is an integer and is at least 3; wherein when m=3, X is uridine or an analogue thereof, and when m>3, at least 3 successive uridines or analogues of uridine occur; n is an integer from 1 to 40, wherein when n=1, G is guanosine or an analogue thereof, when n>1, at least 50% of these nucleotides are guanosine or an analogue thereof; u, v may be independently from each other an integer from 0 to 50; wherein the nucleic acid molecule of formula (I) has a length of at least 50 nucleotides, nucleotides and wherein X.sub.m contains a monotonic uracil sequence having a length of at least 8 uridines or uracil analogues.

2. Nucleic acid according to claim 1, characterized in that the nucleic acid molecule of formula (I) contains a core structure G.sub.lX.sub.mG.sub.n, selected from at least one sequence according to any of SEQ ID NOs: 1-5, 10-18, 23-31, 36-53, 59-66 and 71-79: TABLE-US-00008 (SEQIDNO:1) GGUUUUUUUUUUUUUUUGGG; (SEQIDNO:2) GGGGGUUUUUUUUUUGGGGG; (SEQIDNO:3) GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQIDNO:4) GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU; (SEQIDNO:5) GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU; (SEQIDNO:10) GGGGGGUUUUUUUUGGGGGG; (SEQIDNO:11) GGGGGGUUUUUUUUUGGGGG; (SEQIDNO:12) GGGGGGUUUUUUUUUUGGGG; (SEQIDNO:13) GGGGGUUUUUUUUUUUGGGG; (SEQIDNO:14) GGGGGUUUUUUUUUUUUGGG; (SEQIDNO:15) GGGGUUUUUUUUUUUUUGGG; (SEQIDNO:16) GGGGUUUUUUUUUUUUUUGG; (SEQIDNO:17) GGUUUUUUUUUUUUUUUUGG; (SEQIDNO:18) GUUUUUUUUUUUUUUUUUUG; (SEQIDNO:23) GGGGGGGUUUUUUUUGGGGGGG; (SEQIDNO:24) GGGGGGGUUUUUUUUUGGGGGG; (SEQIDNO:25) GGGGGGGUUUUUUUUUUGGGGG; (SEQIDNO:26) GGGGGGUUUUUUUUUUUGGGGG; (SEQIDNO:27) GGGGGGUUUUUUUUUUUUGGGG; (SEQIDNO:28) GGGGGUUUUUUUUUUUUUGGGG; (SEQIDNO:29) GGGGGUUUUUUUUUUUUUUGGG; (SEQIDNO:30) GGGUUUUUUUUUUUUUUUUGGG; (SEQIDNO:31) GGUUUUUUUUUUUUUUUUUUGG; (SEQIDNO:36) GGGGGGGUUUUUUUUGGGGGGGG; (SEQIDNO:37) GGGGGGGUUUUUUUUUGGGGGGG; (SEQIDNO:38) GGGGGGGUUUUUUUUUUGGGGGG; (SEQIDNO:39) GGGGGGUUUUUUUUUUUGGGGGG; (SEQIDNO:40) GGGGGGUUUUUUUUUUUUGGGGG; (SEQIDNO:41) GGGGGUUUUUUUUUUUUUGGGGG; (SEQIDNO:42) GGGGGUUUUUUUUUUUUUUGGGG; (SEQIDNO:43) GGGUUUUUUUUUUUUUUUUGGGG; (SEQIDNO:44) GGGUUUUUUUUUUUUUUUUUUGGG; (SEQIDNO:45) GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG; (SEQIDNO:46) GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG; (SEQIDNO:47) GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQIDNO:48) GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQIDNO:49) GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG; (SEQIDNO:50) GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQIDNO:51) GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG; (SEQIDNO:52) GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG; (SEQIDNO:53) GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG; (SEQIDNO:59) GGUUUUUUUUGG; (SEQIDNO:60) GGUUUUUUUUUGG; (SEQIDNO:61) GGUUUUUUUUUUGG; (SEQIDNO:62) GGUUUUUUUUUUUGG; (SEQIDNO:63) GGUUUUUUUUUUUUGG; (SEQIDNO:64) GGUUUUUUUUUUUUUGG; (SEQIDNO:65) GGUUUUUUUUUUUUUUGG; (SEQIDNO:66) GGUUUUUUUUUUUUUUUGG; (SEQIDNO:71) GGGUUUUUUUGGG; (SEQIDNO:72) GGGUUUUUUUUGGG; (SEQIDNO:73) GGGUUUUUUUUUGGG; (SEQIDNO:74) GGGUUUUUUUUUUGGG; (SEQIDNO:75) GGGUUUUUUUUUUUGGG; (SEQIDNO:76) GGGUUUUUUUUUUUUGGG; (SEQIDNO:77) GGGUUUUUUUUUUUUUGGG; (SEQIDNO:78) GGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGGUUUUUUUUU UUUUUUGGG;

3. Nucleic acid according to claim 1, characterized in that the nucleic acid comprises the specific formula (II)
poly(X).sub.s(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.apoly(X).sub.t, wherein G, X, N, a, l, m, n, u and v are as defined above for formula (I); and poly(X) is a homopolymeric stretch of nucleic acids, wherein X may be any nucleic acid as defined for X of the nucleic acid molecule according to formula (I); and s, t is each an integer selected, independent from each other, from about 5 to 100, wherein the nucleic acid molecule of formula (II) has a length of at least 50 nucleotides.

4. Nucleic acid according to claim 3, characterized in that the nucleic acid molecule according to formula (II) is specifically a nucleic acid molecule according to formula (IIa):
poly(X)(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a

5. Nucleic acid according to claim 3, characterized in that the nucleic acid molecule according to formula (II) is specifically a nucleic acid molecule according to formula (IIb):
poly(X)(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.apoly(X)

6. Nucleic acid according to claim 3, characterized in that the homopolymeric stretch of nucleic acids poly(X) is a single-stranded, a partially double-stranded or a double-stranded RNA sequence.

7. Nucleic acid according to claim 1, characterized in that the homopolymeric stretch of nucleic acids poly(X) is selected from a single-stranded stretch of cytidines (poly(C)), guanosines (poly(G)), adenosines (poly(A)), uridines (poly(U)), inosines (poly(I)), or from a homopolymeric double-stranded stretch of inosines and cytoines (poly(I:C)), or of adenosines and uridines (poly(A:U)).

8. Nucleic acid according to claim 3, characterized in that the nucleic acid comprises the specific formula (IIIa):
(N.sub.ustem1G.sub.lX.sub.mG.sub.nstem2N.sub.v).sub.a, or the specific formula (IIIb):
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.astem1N.sub.w1stem2N.sub.w2, wherein each G, X, N, a, l, m, n, u and v is as defined above for formula (I); and w1, w2 are integers and independently of each other as defined above for integers u, v of formula (I); and stem1, stem2 are palindromic sequences and form a stem loop structure, wherein the palindromic sequences are each formed by a nucleic acid sequence having a length of about 5 to 50 nucleic acids, selected from adenosine, guanosine, cytidine, uridine, thymidine, or an anologue thereof.

9. Nucleic acid according to claim 1, characterized in that the nucleic acid molecule of formula (I), (II), (IIa), (IIb), (IIIa) and/or (IIIb) is in the form of RNA or DNA, is single-stranded, double-stranded or partially double-stranded, and/or is linear or circular.

10. Nucleic acid according to claim 1, characterized in that the nucleic acid of any of formula (I), (II), (IIa), (IIb), (IIIa) and/or (IIIb) is in the form of a single-stranded RNA, a double-stranded RNA or a or a partially double-stranded RNA, preferably a partially double-stranded RNA.

11. Nucleic acid according to claim 1, characterized in that the nucleic acid of any of formula (I), (II), (IIa), (IIb), (IIIa) and/or (IIIb) is present in the form of RNA and additionally has at the 5 terminus a Cap structure and/or at the 3 terminus a poly-A tail and/or at the 3 terminus a poly-C tail.

12. Nucleic acid according to claim 1, characterized in that the nucleic acid is prepared by an in vitro translation.

13. An immunostimulating agent comprising a nucleic acid of claim 1.

14. Pharmaceutical composition containing a nucleic acid according to claim 1, a pharmaceutically acceptable carrier and, optionally, further auxiliary substances, additives and/or adjuvants.

15. Pharmaceutical composition according to claim 14, additionally comprising at least one pharmaceutically active component.

16. Pharmaceutical composition according to claim 15, characterized in that the at least one pharmaceutically active component is selected from the group consisting of peptides, proteins, nucleic acids, (therapeutically active) low molecular weight organic or inorganic compounds having a molecular weight less than 5000, sugars, antigens, antibodies, pathogens, attenuated pathogens, de-activated pathogens, (human) cells, cellular fragments or fractions and other therapeutic agents, which are preferably adapted to exhibit enhanced transfection properties, including by complexation with lipids and/or polycationic compounds, including polycationis peptides.

17. Pharmaceutical composition according to claim 1, characterized in that the composition contains at least one further adjuvant, which is an immunostimulating agent, selected from the group consisting of cationic peptides, including polypeptides including protamine, nucleoline, spermine or spermidine, cationic polysaccharides, including chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE (propanediamine); BAY R1005 ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoyl-amide hydroacetate); CALCITRIOL (1-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAP (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i) N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine); imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-gamma; interleukin-1beta; interleukin-2; interleukin-7; interleukin-12; ISCOMS (Immunostimulating Complexes); ISCOPREP 7.0.3.; liposomes; LOXORIBINE (7-allyl-8-oxoguanosine (guanine)); LT oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59; (squalene-water emulsion); MONTANIDE ISA 51 (purified incomplete Freund's adjuvant); MONTANIDE ISA720 (metabolisable oil adjuvant); MPL (3-Q-desacyl-4-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))ethylamide, monosodium salt); MURAMETIDE (Nac-Mur-L-Ala-D-Gln-OCH.sub.3); MURAPALMITINE and D-MURAPALMITINE (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN (beta-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres/nanospheres); PLURONIC L121; PMMA (polymethyl methacrylate); PODDS (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol); SAF-1 (Syntex adjuvant formulation); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane (2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine (octadecyltyrosine hydrochloride); Theramid (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide or [thr 1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju-phos, Alhydrogel, Rehydragel, etc.; emulsions, such as CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin, etc.; copolymers, such as Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, such as Stealth, etc., cochleates, such as BIORAL, etc.; plant derived adjuvants, such as QS21, Quil A, Iscomatrix, ISCOM, etc.; preferred adjuvants suitable for costimulation may include e.g. Tomatine, biopolymers, such as PLG, PMM, Inulin, etc.; microbe derived adjuvants, such as Romurtide, DETOX, MPL, CWS, Mannose, CpG7909, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP, etc.; preferred adjuvants suitable as antagonists may e.g. include CGRP neuropeptide, or from cationic or polycationic compounds which are suitable for depot and delivery, including protamine, nucleoline, spermin or spermidine, or other cationic peptides or proteins, including 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 Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones. Additionally, preferred cationic or polycationic proteins or peptides may be selected from following proteins or peptides having the following total formula: (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x, wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide, cationic polysaccharides, for example chitosan, polybrene, cationic polymers, including polyethyleneimine (PEI), cationic lipids, including DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, 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-(-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIPS: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, including modified polyaminoacids, including -aminoacid-polymers or reversed polyamides, modified polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridinium bromide)) modified acrylates, including pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified Amidoamines including pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE), including diamine end modified 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymers dendrimers, including polypropylamine dendrimers or pAMAM based dendrimers, polyimine(s), including PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar backbone based polymers, including cyclodextrin based polymers, dextran based polymers, Chitosan, silan backbone based polymers, including PMOXA-PDMS copolymers, blockpolymers consisting of a combination of one or more cationic blocks (including selected og a cationic polymer as mentioned above) and of one or more hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); or may be selected from nucleic acids of formula (IV): G.sub.lX.sub.mG.sub.n, wherein: G is guanosine, uridine or an analogue of guanosine or uridine; X is guanosine, uridine, adenosine, thymidine, cytidine or an analogue of the above-mentioned nucleotides; l is an integer from 1 to 40, wherein when l=1 G is guanosine or an analogue thereof, when l>1 at least 50% of the nucleotides are guanosine or an analogue thereof; m is an integer and is at least 3; wherein when m=3 X is uridine or an analogue thereof, when m>3 at least 3 successive uridines or analogues of uridine occur; n is an integer from 1 to 40, wherein when n=1 G is guanosine or an analogue thereof, when n>1 at least 50% of the nucleotides are guanosine or an analogue thereof; or from nucleic acids of formula (V): C.sub.lX.sub.mC.sub.n, wherein: C is cytidine, uridine or an analogue of cytidine or uridine; X is guanosine, uridine, adenosine, thymidine, cytidine or an analogue of the above-mentioned nucleotides; l is an integer from 1 to 40, wherein when l=1 C is cytidine or an analogue thereof, when l>1 at least 50% of the nucleotides are cytidine or an analogue thereof; m is an integer and is at least 3; wherein when m=3 X is uridine or an analogue thereof, when m>3 at least 3 successive uridines or analogues of uridine occur; n is an integer from 1 to 40, wherein when n=1 C is cytidine or an analogue thereof, when n>1 at least 50% of the nucleotides are cytidine or an analogue thereof.

18. Pharmaceutical composition according to claim 14 to 17, characterized in that the pharmaceutical composition is a vaccine.

19-23. (canceled)

24. Kit containing a nucleic acid according to claim 1, and also optionally technical instructions for use with information on the administration and dosage of the nucleic acid or the pharmaceutical composition.

25. Method of treating a disorder or disease selected from the group consisting of cancer disease, infectious disease, autoimmune disease and allergy by administering to a patient in need thereof a pharmaceutically effective amount of a nucleic acid according to claim 1.

Description

FIGURES

[0194] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0195] FIG. 1: shows the TNF inducing capacity of DOTAP formulated RNAs according to formula (I). PBMCs were seeded at a density of 2*10.sup.5/well/200 l Medium and stimulated with RNA (4 g/ml) formulated with DOTAP (12 g/ml) for 20 h. A TNF-ELISA was then performed with cell free supernatants. As can be seen in FIG. 1, secretion of TNF is significantly induced by the inventive nucleic acids according to formula (I), particularly by mRNA sequences according to SEQ ID NOs: 114 to 119 inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ ID NO: 114 (R820/(N100).sub.2), SEQ ID NO: 115 (R719/(N100).sub.5), SEQ ID NO: 116 (R720/(N100).sub.10), SEQ ID NO: 117 (R821/(N40T20N40).sub.2), SEQ ID NO: 118 (R722/(N40T20N40).sub.5), and SEQ ID NO: 119 (R723/(N40T20N40).sub.10)) and controls G.sub.2U.sub.20G.sub.2 (GGUUUUUUUUUUUUUUUUUUUUGG) (SEQ ID NO: 125), Seq. U.sub.21: UUUUUUUUUUUUUUUUUUUUU (SEQ ID NO: 126) (Phosphodiester) and Poly(U) (Sigma, 803-1000 kDa).

[0196] FIG. 2: shows the IFN inducing capacity of DOTAP formulated RNAs according to formula (I). PBMCs were seeded at a density of 2*10.sup.5/well/200 l Medium and stimulated with RNA (2 g/ml) formulated with DOTAP (12 g/ml) for 20 h. An IFN-ELISA was then performed with cell free supernatants. As can be seen in FIG. 2, secretion of IFN is significantly induced by the inventive nucleic acids according to formula (I), particularly by mRNA sequences according to SEQ ID NOs: 114 to 119 inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ ID NO: 114 (R820/(N100).sub.2), SEQ ID NO: 115 (R719/(N100).sub.5), SEQ ID NO: 116 (R720/(N100).sub.10), SEQ ID NO: 117 (R821/(N40T20N40).sub.2), SEQ ID NO: 118 (R722/(N40T20N40).sub.5), and SEQ ID NO: 119 (R723/(N40T20N40).sub.10)) and controls G.sub.2U.sub.20G.sub.2 (GGUUUUUUUUUUUUUUUUUUUUGG) (SEQ ID NO: 125), Seq. U.sub.21: UUUUUUUUUUUUUUUUUUUUU (SEQ ID NO: 126) (Phosphodiester) and Poly(U) (Sigma, 800-1000 kDa).

EXAMPLES

[0197] The following Examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

1. Synthesis of Exemplary Nucleic Acids of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the Invention

[0198] RNA oligonucleotides, as examples of the nucleic acid of the general formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention, were prepared by automatic solid-phase synthesis by means of phosphoramidite chemistry (including sequences according to SEQ ID NOs: 84-85 (formula (I)), SEQ ID NOs: 86-87 (formula (Ia)), SEQ ID NOs: 88-94 (formulas (II), (IIa) and (IIb)), and SEQ ID NOs: 107-108 (formulas (IIIa) and (IIIb))). In each case the RNA-specific 2-hydroxyl groups of the nucleotides were protected with TBDMS protecting groups. In the synthesis of phosphorothioates, Beaucage reagent was used for the oxidation. The cleavage of carrier material and of the base-labile protecting groups was carried out with methylamine, and the cleavage of the TBDMS protecting group was effected with triethylamine hydrofluoride.

[0199] The crude product was purified by means of HPLC either by ion-pair chromatography, by ion-exchange chromatography or by a combination of the two methods, desalinated and dried. The product was checked for purity and correct base composition by mass spectrometry.

[0200] According to an alternative way, the above sequences were prepared by in vitro translation based on DNA vectors or oligonucleotide sequences carrying the inventive sequences.

2. In Vitro Immunostimulation with Exemplary Nucleic Acids of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the Invention [0201] a) For the stimulation of mouse BDMCs (bone marrow derived dendritic cells), 3 l of oligofectamine were mixed with 30 l of FCS-free IMDM medium (BioWhittaker, catalogue no. BE12-722F) and incubated at room temperature for 5 minutes. 6 g of a nucleic acid according to SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid forming a single experiment), respectively, in the form of RNA, was mixed with 60 l of FCS-free IMDM and mixed with oligofectamine/IMDM, and incubated for 20 minutes at room temperature. 33 l of this mixture were then placed for cultivation overnight in a well of a 96-well microtitre culture plate which contained 200,000 mouse BDMCs in 200 l of FCS-free IMDM medium. After 4 hours, 100 l of IMDM containing 20% FCS were added and, after 16 hours' co-incubation, the supernatant was removed and tested for interleukin-6 (IL-6) and interleukin-12 (IL-12) by a cytokine ELISA. Comparison tests were carried out analogously to the above sequences using the immunostimulating uncapped wild-type mRNA of beta-galactosidase (lacZ), complexed with protamine. [0202] It was possible to show that the nucleic acids of formulas (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention, present in the form of RNA, in particular the sequences according to the invention of SEQ ID NOs: 84-94 and 107-108, have good immunostimulating properties for stimulation of an innate immune response. [0203] b) Human PBMCs were obtained via a Ficoll density gradient and cultivation overnight in X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q), which contained 1% glutamine and 1% penicillin in the presence of 10 g/ml of the nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention in the form of RNA, in particular of the sequences according to the invention of SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid forming a single experiment). [0204] For stimulation, 3 l of oligofectamine were mixed with 30 l of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q) and incubated at room temperature for 5 minutes. 6 g of the nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention in the form of RNA, in particular the sequences according to the invention SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid in a single experiment), respectively, were mixed with 60 l of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q) and, mixed with oligofectamine/X-VIVO medium, incubated for 20 minutes at room temperature. 33 l of this mixture were then placed for cultivation overnight in a well of a 96-well microtitre culture plate which contained 200,000 PBMCs in 200 in of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q). After co-incubation for 16 hours, the supernatant was removed and tested for interleukin-6 (IL-6) and interleukin-12 (IL-12) and TNF by means of a cytokine-ELISA. Comparison tests were carried out analogously to the sequences according to the invention (see above) with the immunostimulating oligo RNA40 (5-GCCCGUCUGUUGUGUGACUC-3, SEQ ID NO: 113). [0205] It was possible to show that the inventive nucleic acids in the form of RNA, in particular having the sequences according to the invention either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention as defined above have good immunostimulating properties.
3. In Vivo Immunostimulation with Exemplary Nucleic Acids of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the InventionUse as Adjuvant

[0206] BALB/c mice (5 per group) were injected with beta-galactosidase protein and with an adjuvant (as defined herein) on days 0 and 10. The mice were sacrificed on day 20 and the blood serum was used for an antibody test against beta-galactosidase protein by means of ELISA, and the IL-6, IL-12 and TNF-alpha values were determined analogously to the above-described in vitro cultures.

4. Stimulation of Human Cells with an Adjuvant According to the Invention in the Form of a Nucleic Acid Molecule of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) [0207] a) In order to determine the immunogenic activity of nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention as defined above in the form of adjuvants, particularly of nucleic acids containing a sequence according to SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid again forming a single experiment) were co-incubated with human cells. To this end, human PBMC cells, for example, were co-incubated for 16 hours in X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q), enriched with 2 mM L-glutamine (BioWhittaker), 10 U/ml penicillin (BioWhittaker) and 10 g/ml streptomycin, with 10 g/ml of RNA (mRNA coding for -galactosidase and optionally with 10 g/ml protamine. The supernatants were removed and the release of IL-6 and TNFalpha was analysed by means of ELISA. [0208] b) In a further experiment, the release of TNF-alpha by human PBMC cells was determined after stimulation with inventive nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention (SEQ ID NOs: 84-94 and 107-108, each type of nucleic acid in a single experiment, see above) and also adjuvants used according to the invention. [0209] To that end, human PBMC cells were co-incubated for 16 hours with 10 g/ml said inventive nucleic acids in X-VIVO 15 medium (BioWhittaker), enriched with 2 mM L-glutamine (BioWhittaker), 10 U/ml penicillin (BioWhittaker) and 10 g/ml streptomycin. The supernatants were removed and analysed by means of ELISA.

5. Secretion of TNF and IFN- in Human PBMCs

[0210] For this experiments, several inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119, were formulated with DOTAP (Roche).

[0211] The inventive nucleic acid sequences used in the experiment were [0212] SEQ ID NO: 114 (R820/(N100).sub.2); [0213] SEQ ID NO: 115 (R719/(N100).sub.5); [0214] SEQ ID NO: 116 (R720/(N100).sub.10); [0215] SEQ ID NO: 117 (R821/(N40T20N40).sub.2); [0216] SEQ ID NO: 118 (R722/(N40T20N40).sub.5); and [0217] SEQ ID NO: 119 (R723/(N40T20N40).sub.10).

[0218] Human PBMCs were then stimulated with the formulated RNAs at a concentration of 8 g/ml and 12 g/ml DOTAP for 20 hours. The Supernatants were then investigated for the secretion of TNFa and IFN-a using a matched-paired ELISA.

[0219] For the experiment, human PBMCs were obtained via a Ficoll density gradient and cultivation for 20 hours in X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q), which contained 1% glutamine and 1% penicillin in the presence of 2 or 4 g/ml of the above nucleic acids for IFN or TNF stimulation respectively. For formulation and stimulation, 3 or 6 g RNA in HBS buffer were transferred to a vial containing 18 g N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammonium methylsulfate (DOTAP) (Roche Diagnostics, catalogue no. 11 811 177 001) in HBS buffer and carefully mixed by gently pipetting the mixture several times. The transfection mixture was incubated for 15 min at 15-25 C. 1 volume of the DOTAP/nucleic acid mixture was then gently diluted with 7.3 volumes of X-Vivo medium. 100 l of this mixture were then placed for cultivation overnight in a well of a 96-well microtitre culture plate which contained 2*10.sup.5 PBMCs in 100 l of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q). After coincubation for 20 hours, the supernatant was removed and tested for IFN and TNF by means of a cytokine-ELISA. Comparison tests were carried out analogously to the sequences according to the invention (see above) with the immunostimulating oligo G.sub.2U.sub.20G.sub.2 (Phosphothioat-modifed), Poly(U) (Sigma, Taufkirchen, Germany) and the oligo U.sub.21 (Phophodiester).

[0220] The results are shown in FIGS. 1 and 2. FIG. 1 shows the TNF inducing capacity of DOTAP formulated RNAs. PBMCs were seeded at a density of 2*10.sup.5/well/200 l Medium and stimulated with RNA (4 g/ml) formulated with DOTAP (12 g/ml) for 20 h. A TNF-ELISA was then performed with cell free supernatants. FIG. 2 shows the IFN inducing capacity of DOTAP formulated RNAs. PBMCs were seeded at a density of 2*10.sup.5/well/200 l Medium and stimulated with RNA (2 g/ml) formulated with DOTAP (12 g/ml) for 20 h. An IFN-ELISA was then performed with cell free supernatants.

[0221] As can be seen in FIG. 1 and FIG. 2, both secretion of TNF and IFN is significantly induced by the inventive nucleic acids according to formula (I), particularly by mRNA sequences according to SEQ ID NOs: 114 to 119 inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ ID NO: 114 (R820/(N100).sub.2), SEQ ID NO: 115 (R719/(N100).sub.5), SEQ ID NO: 116 (R720/(N100).sub.10), SEQ ID NO: 117 (R821/(N40T20N40).sub.2), SEQ ID NO: 118 (R722/(N40T20N40).sub.5), and SEQ ID NO: 119 (R723/(N40T20N40).sub.10)) versus control sequences G.sub.2U.sub.20G.sub.2 (Phosphothioat-modifed), Poly(U) (Sigma, Taufkirchen, Germany) and the oligo U.sub.21 (Phophodiester).

ADVANTAGES OF THE INVENTION

[0222] A nucleic acid of the general formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention may be used as immunostimulating agent as such for stimulating the innate immune system of a patient to be treated. This immunstimulating property may well be enhanced by the addition of other compounds known in the art as actively stimulating the innate immune response to the inventive nucleic acids, e.g. by lipid modification or addition of additional adjuvants. The inventive nucleic acids as defined herein, particularly those according to formula (I) comprising the structure (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v), or of derivatives thereof, exhibit a significant better amplification in bacteria, e.g. E. coli. It is furthermore particularly advantageous, if the inventive nucleic acid (N.sub.vG.sub.lX.sub.mG.sub.nN.sub.u).sub.a of formula (I), or of derivatives thereof, is a partially double-stranded nucleic acid molecule or a mixture of a single-stranded and a double-stranded nucleic acid molecule, since such a (partially double-stranded) inventive nucleic acid molecule according to formula (I) (or of formula (Ia), (II) (IIa), (IIb), (IIIa) and/or (IIIb)), can positively stimulate the innate immune response in a patient to be treated by addressing the PAMP-(pathogen associated molecular pattern) receptors for single-stranded RNA (TLR-7 and TLR-8) as well as the PAMP-receptors for double-stranded RNA (TLR-3, RIG-I and MDA-5). Receptors TLR-3, TLR-7 and TLR-8 are located in the endosome and are activated by RNA taken up by the endosome. In contrast, RIG-I and MDA-5 are cytoplasmic receptors, which are activated by RNA which was directly taken up into the cytoplasm or which has been released from the endosomes (endosomal release or endosomal escape). Accordingly, a partially double-stranded inventive nucleic acid (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a of formula (I) (or of derivatives thereof, e.g. (a partially double-stranded) inventive nucleic acid molecule according to formula (Ia), (II) (IIa), (IIb), (IIIa) and (IIIb) as defined herein)) is capable of activating different signal cascades of immunostimulation and thus leads to an increased innate immune response or enhances such a response significantly. A further advantage of the invention is the high induction of the antiviral cytokine IFNalpha which is preferred in stimulation of the innate immune system. An often underestimated limitation of generally accepted immunostimulating nucleic acids (e.g. poly A:U and poly I:C) is the undefined structure of them which results in regulatory restrictions.