METHODS OF IMMUNOSTIMULATION WITH COMPLEXES OF RNA AND CATIONIC CARRIERS

Abstract

The present invention relates to a complexed RNA, comprising at least one RNA complexed with one or more oligopeptides, wherein the oligopeptide has a length of 8 to 15 amino acids and has the empirical formula (Arg).sub.l;(;Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x. The invention further relates to a method for transfecting a cell or an organism, thereby applying the inventive complexed RNA. Additionally, pharmaceutical compositions and kits comprising the inventive complexed RNA, as well as the use of the inventive complexed RNA for transfecting a cell, tissue or an organism and/or for modulating, preferably inducing or enhancing, an immune response are disclosed herein.

Claims

1. A method of stimulating an immune response in a subject comprising administering an effective amount of a pharmaceutical composition comprising a purified mRNA encoding a coronavirus antigen to the subject, wherein said mRNA is complexed or formulated with a cationic carrier molecule.

2. The method of claim 1, wherein the coronavirus antigen is from a SARS-associated coronavirus.

3. The method of claim 1, wherein the coronavirus antigen is from human coronavirus 229E or human coronavirus Oc43.

4. The method of claim 1, further defined as a method for preventing disease from an infection with a SARS-associated coronavirus.

5. The method of claim 1, wherein the administering is by injection.

6. The method of claim 5, wherein the administering is by intramuscular injection.

7. The method of claim 1, wherein the cationic carrier comprises a cationic polymer or cationic polypeptide.

8. The method of claim 1, wherein the cationic carrier comprises a cationic lipid.

9. The method of claim 1, wherein the mRNA is complexed with a cationic carrier and forms nanoplexes, lipoplexes or polyplexes.

10. The method of claim 1, wherein the mRNA is complexed with a cationic carrier and forms lipoplexes.

11. The method of claim 1, wherein the mRNA comprises a 5 cap structure.

12. The method of claim 1, wherein the mRNA comprises a poly-A tail of 10 to 200 adenosine nucleotides.

13. The method of claim 1, wherein the mRNA comprises a poly-C tail of 10 to 200 cytosine nucleotides.

14. The method of claim 1, wherein the mRNA comprises a 5 and/or a 3 untranslated region (UTR).

15. The method of claim 1, wherein the mRNA comprises a G/C content in the antigen coding sequence which is greater than the G/C content of a coding region of the native mRNA sequence encoding the antigen.

16. The method of claim 1, wherein the mRNA comprises an antigen coding sequence that is modified, compared with a native RNA encoding the antigen, such that at least one codon of the native mRNA 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.

17. The method of claim 1, wherein the mRNA has been modified by introduction of a non-native nucleotide compared with a corresponding native mRNA nucleotide and/or by covalent coupling of the mRNA with a further chemical moiety.

18. The method of claim 17, wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA.

19. The method of claim 17, wherein the mRNA comprises at least one nucleotide that is substituted with a nucleotide analog selected from the group 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, 6-chloropurineriboside-5-triphosphate, 7-deazaadenosine-5-triphosphate, 7-deazaguanosine-5-triphosphate, 8-azaadenosine-5 -triphosphate, 8-azidoadenosine-5-triphosphate, benzimidazole-riboside-5-triphosphate, N1-methyladenosine-5-triphosphate, N1-methylguanosine-5-triphosphate, N6-methyladenosine-5-triphosphate, 06-methylguanosine-5-triphosphate, pseudouridine-5-triphosphate, or puromycin-5-triphosphate, xanthosine-5-triphosphate.

20. The method of claim 1, wherein the mRNA has been purified by chromatography.

21. The method of claim 20, wherein the mRNA has been purified by HPLC.

22. The method of claim 21, wherein the mRNA has been purified by reverse phase (RP) HPLC.

23. The method of claim 1, wherein the composition comprises polyethylene glycol (PEG).

24. The method of claim 1, further defined as a method of stimulating antibody production in the subject.

25. The method of claim 1, further defined as a method for stimulating a CD4+ and/or a CD8+ T-cell response.

Description

FIGURES

[0245] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0246] FIG. 1: depicts the sequence of a stabilized luciferase mRNA sequence, wherein the native luciferase encoding mRNA is modified with a poly-A/poly-C-tag (A70-C30). This first construct (construct CAP-Ppluc(wt)-muag-A70-C30, SEQ ID NO: 35) contained following sequence elements:

[0247] stabilizing sequences from the alpha-Globin gene,

[0248] 70Adenosin at the 3-terminal end (poly-A-tail),

[0249] 30Cytosin at the 3-terminal end (poly-C-tail);

[0250] represented by following symbols:

[0251] ______=coding sequence

[0252] .sub.==3-UTR of the alpha globin gene

[0253] . . . =poly-A-tail

[0254] .sub.- - - =poly-C-tail

[0255] FIG. 2: shows the sequence of a stabilized luciferase mRNA sequence, wherein the construct according to SEQ ID NO: 35 (see FIG. 1) is further modified with a GC-optimized sequence for a better codon usage. The final construct (construct CAP-Ppluc(GC)-muag-A70-C30, SEQ ID NO: 36) contained following sequence elements:

[0256] GC-optimized sequence for a better codon usage

[0257] stabilizing sequences from the alpha-Globin gene

[0258] 70Adenosin at the 3-terminal end (poly-A-tail),

[0259] 30Cytosin at the 3-terminal end (poly-C-tail);

[0260] represented by following symbols:

[0261] ______=coding sequence

[0262] .sub.==modified 3-UTR of the alpha globin gene

[0263] . . . =poly-A-tail

[0264] .sub.- - - =poly-C-tail

[0265] FIG. 3: shows the coding sequence of the sequence according to SEQ ID NO: 35 (SEQ ID NO: 37) (see FIG. 1).

[0266] FIG. 4: shows the GC-optimized coding sequence of the sequence according to SEQ ID NO: 36 (SEQ ID NO: 38) (see FIG. 2). The GC-optimized codons are underlined.

[0267] FIG. 5: shows the immunostimulatory effect of RNA complexed with nona-arginine ((Arg).sub.9) in hPBMC cells by measuring IL-6 production. As can be seen, hPBMC cells show a significant IL-6 production, i.e. a significant immunostimulatory effect of RNA complexed with nona-arginine ((Arg).sub.9).

[0268] FIG. 6: shows the immunostimulatory effect of RNA complexed with nona-arginine ((Arg).sub.9) in hPBMC cells by measuring TNF-alpha production. As can be seen, hPBMC cells show a significant TNF-alpha production, i.e. a significant immunostimulatory effect of RNA complexed with nona-arginine ((Arg).sub.9).

[0269] FIG. 7: shows in an comparative example the comparison of immunostimulatory effects of RNA complexed with either nona-arginine ((Arg).sub.9) or poly-L-arginine, respectively, in hPBMCs. Advantageously, a significant immunostimulatory effect can be observed for mass ratios lower than 1:5 (RNA:nona-arginine) (1:10; 1:8; 1:5; 1:2; 1:1; 2:1). However, when using mass ratios of RNA:nona-arginine (5:1) no significant TNFalpha production can be observed. The same applies to stimulation experiments, using nona-arginine ((Arg).sub.9) or mRNA alone. Additionally, it was observed, that complexation of mRNA with poly-L-arginine leads to significantly lower induction of TNF-alpha production in comparison to nona-arginine ((Arg).sub.9). Apparently, higher concentrations of poly-L-arginine appear to be toxic for cells transfected therewith, particularly when using a mass ratio of 1:2 RNA:poly-L-arginine:RNA or higher, since the cells were lysed.

[0270] FIG. 8: shows luciferase expression upon transfection of complexes of RNA with nona-arginine ((Arg).sub.9) in HeLa cells. As may be derived from FIG. 8 a mass ratio of less than 2:1 (RNA:nona-arginine) appears to be advantageous. In contrast, complexation with (high molecular mass) poly-L-arginine does not lead to a significant luciferase-activity. Thus, (high molecular mass) poly-L-arginine does not appear to be suitable for transfection of mRNA.

[0271] FIG. 9: depicts in a comparative example the luciferase expression upon transfection of complexes of RNA with hepta-arginine ((Arg).sub.7) in HeLa cells. As may be derived from FIG. 9, transfection of complexes of RNA with hepta-arginine ((Arg).sub.7) does not lead to a significant luciferase-activity. Thus, hepta-arginine ((Arg).sub.7) does also not appear to be suitable for transfection of mRNA.

[0272] FIG. 10: shows the immunostimulatory effect of RNA complexed with hepta-arginine ((Arg).sub.7) in hPBMC cells by measuring IL-6 production. As can be seen, hPBMC cells show a significant IL-6 production, i.e. a significant immunostimulatory effect of RNA complexed with hepta-arginine ((Arg).sub.7).

[0273] FIG. 11: shows the immunostimulatory effect of RNA complexed with hepta-arginine ((Arg).sub.7) in hPBMC cells by measuring TNF-alpha production. As can be seen, hPBMC cells show a significant TNF-alpha production, i.e. a significant immunostimulatory effect of RNA complexed with hepta-arginine ((Arg).sub.7).

[0274] FIG. 12: shows the effect of RNA complexed with R9 peptide on the expression of luciferase in HeLa cells.

[0275] FIG. 13: shows the effect of RNA complexed with R9H3 peptide on the expression of luciferase in HeLa cells.

[0276] FIG. 14: shows the effect of RNA complexed with H3R9H3 peptide on the expression of luciferase in HeLa cells.

[0277] FIG. 15: shows the effect of RNA complexed with YYYR9SSY (SEQ ID NO: 401) peptide on the expression of luciferase in HeLa cells.

[0278] FIG. 16: shows the effect of RNA complexed with H3R9SSY peptide on the expression of luciferase in HeLa cells.

[0279] FIG. 17: shows the effect of RNA complexed with (RKH)4 peptide on the expression of luciferase in HeLa cells.

[0280] FIG. 18: shows the effect of RNA complexed with Y(RKH)2R peptide on the expression of luciferase in HeLa cells.

[0281] FIG. 19: shows the effect of Histidin in terminal positions on the transfection efficiacy.

[0282] FIG. 20: shows the effect of neutral amino acids in terminal positions on the transfection efficiacy.

[0283] FIG. 21: shows the immunostimulatory effect of RNA complexed with R9H3 on secretion of TNFalpha in hPBMCs.

[0284] FIG. 22: shows the immunostimulatory effect of RNA complexed with R9H3 on secretion of IL-6 in hPBMCs.

EXAMPLES

[0285] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

Example 1Preparation of Luciferase mRNA Constructs

[0286] In the following experiments a stabilized luciferase mRNA sequence was prepared and used for transfection experiments, wherein the native luciferase encoding mRNA was modified with a poly-A/poly-C-tag (A70-C30) and was GC-optimized for a better codon-usage and further stabilized.

[0287] A first construct (construct CAP-Ppluc(wt)-muag-A70-C30, SEQ ID NO: 35) contained following sequence elements: [0288] stabilizing sequences from the alpha-Globin gene [0289] 70 (Adenosin at the 3-terminal end [0290] 30 (Cytosin at the 3-terminal end

[0291] The final construct (construct CAP-Ppluc(GC)-muag-A70-C30, SEQ ID NO: 36), as used herein for the following experiments, contained following sequence elements: [0292] GC-optimized sequence for a better codon usage [0293] stabilizing sequences from the alpha-Globin gene [0294] 70 (Adenosin at the 3-terminal end [0295] 30 (Cytosin at the 3-terminal end

[0296] These sequences are also shown in FIGS. 1 and 2 (SEQ ID NOs: 35 and 36). The respective coding sequences are shown in FIGS. 3 and 4 (SEQ ID NOs: 35 and 36)

Example 2In Vitro Transcription of Stabilized Luciferase mRNA

[0297] The stabilized luciferase mRNA according to SEQ ID NO: 35 or 36 (Luc-RNActive) was transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Deutschland) following the manufactures instructions.

[0298] All mRNA-transcripts contained a 70 bases poly-A-tail and a 5-Cap-structure. The 5-Cap-structure was obtained by adding an excess of N7-Methyl-Guanosin-5-Triphosphat-5-Guanosin.

Example 3Forming a Complex of RNA with Nona-Arginine ((Arg).SUB.9.), Poly-L-Arginine or Further Peptides Based on (Arg).SUB.9., Respectively

[0299] 15 g RNA stabilized luciferase mRNA according to SEQ ID NO: 36 (Luc-RNActive) were mixed in different mass ratios with nona-arginine (Arg.sub.9) or poly-L-arginine (Sigma-Aldrich; P4663; 5000-15000 g/mol), thereby forming a complex. Following mass ratios were used as shown exemplarily for ((Arg).sub.9). Poly-L-arginine was used for comparative examples following the same instructions.

TABLE-US-00004 (Arg).sub.9 (Arg).sub.9 RNA (Arg).sub.9 H.sub.20 Concentration Ratio RNA (Arg).sub.9 g g l l l (Arg).sub.9 [M] (Arg).sub.9/RNA 1 Mock 70.0 0 2 (Arg).sub.9 alone 150 3 67.0 151.32 3 RNA alone 15 3.8 66.3 0.00 4 1 10 15 150.0 3.8 3.0 63.3 151.32 10:1 5 1 8 15 120.0 3.8 2.4 63.9 121.06 8:1 6 1 5 15 75.0 3.8 1.5 64.8 75.66 5:1 7 1 2 15 30.0 3.8 0.6 65.7 30.26 2:1 8 1 1 15 15.0 3.8 15.0 51.3 15.13 1:1 9 2 1 15 7.5 3.8 7.5 58.8 7.57 1:2 10 5 1 15 3.0 3.8 3.0 63.3 3.03 1:5 11 8 1 15 1.9 3.8 1.9 64.4 1.89 1:8 12 10 1 15 1.5 3.8 1.5 64.8 1.51 1:10

[0300] Additionally, further complexed RNAs based on (Arg).sub.9 were prepared above using the following peptides for complexation:

TABLE-US-00005 R9: (SEQIDNO:2) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg R9H3: (SEQIDNO:39) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-His H3R9H3: (SEQIDNO:40) His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- His-His-His YSSR9SSY: (SEQIDNO:41) Tyr-Ser-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr H3R9SSY: (SEQIDNO:42) His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr (RKH)4: (SEQIDNO:43) Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His Y(RKH)2R: (SEQIDNO:44) Tyr-Arg-Lys-His-Arg-Lys-His-Arg

[0301] For complexation, 4 g stabilized luciferase mRNA according to SEQ ID NO: 36 (Luc-RNActive) were mixed in molar ratios with the respectively peptide (according to formula I), thereby forming a complex. Afterwards the resulting solution was adjusted with water to a final volume of 50 l and incubated for 30 minutes at room temperature. The used ratios are indicated in the tables given below. HeLa-cells (15010.sup.3/well) were then seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out.

TABLE-US-00006 R9: Formulation > N/P R9 Molar ratio Mass ratio RNA R9 RNA g Peptid g N/P 1.00 10000.00 1.00 23.72 50.00 1.00 5000.00 1.00 11.86 25.00 1.00 2500.00 1.00 5.93 12.50 1.00 1000.00 1.00 2.37 5.00 1.00 500.00 1.00 1.19 2.50 1.00 100.00 1.00 0.24 0.50 1.00 10.00 1.00 0.02 0.05 R9H3: Formulation > N/P R9H3 Molar ratio Mass ratio RNA R9H3 RNA g Peptid g N/P 1.00 10000.00 1.00 30.58 50.00 1.00 5000.00 1.00 15.29 25.00 1.00 2500.00 1.00 7.65 12.50 1.00 1000.00 1.00 3.06 5.00 1.00 500.00 1.00 1.53 2.50 1.00 100.00 1.00 0.31 0.50 1.00 10.00 1.00 0.03 0.05 H3R9H3: Formulation > N/P H3R9H3 Molar ratio Mass ratio RNA H3R9H3 RNA g Peptid g N/P 1.00 10000.00 1.00 37.43 50.00 1.00 5000.00 1.00 18.72 25.00 1.00 2500.00 1.00 9.36 12.50 1.00 1000.00 1.00 3.74 5.00 1.00 500.00 1.00 1.87 2.50 1.00 100.00 1.00 0.37 0.50 1.00 10.00 1.00 0.04 0.05 YSSR9SSY: Formulation > N/P YSSR9SSY Molar ratio Mass ratio RNA YSSR9SSY RNA g Peptid g N/P 1.00 10000.00 1.00 34.95 50.00 1.00 5000.00 1.00 17.48 25.00 1.00 2500.00 1.00 8.74 12.50 1.00 1000.00 1.00 3.50 5.00 1.00 500.00 1.00 1.75 2.50 1.00 100.00 1.00 0.35 0.50 1.00 10.00 1.00 0.03 0.05 H3R9SSY: Formulation > N/P H3R9SSY Molar ratio Mass ratio RNA H3R9SSY RNA g Peptid g N/P 1.00 10000.00 1.00 36.18 50.00 1.00 5000.00 1.00 18.09 25.00 1.00 2500.00 1.00 9.05 12.50 1.00 1000.00 1.00 3.62 5.00 1.00 500.00 1.00 1.81 2.50 1.00 100.00 1.00 0.36 0.50 1.00 10.00 1.00 0.04 0.05 (RKH)4: Formulation > N/P (RKH)4 Molar ratio Mass ratio RNA (RKH)4 RNA g Peptid g N/P 1.00 10000.00 1.00 28.38 44.44 1.00 5000.00 1.00 14.19 22.22 1.00 2500.00 1.00 7.10 11.11 1.00 1000.00 1.00 2.84 4.44 1.00 500.00 1.00 1.42 2.22 1.00 100.00 1.00 0.28 0.44 1.00 10.00 1.00 0.03 0.04 Y(RKH)2R: Formulation > N/P Y(RKH)2R Molar ratio Mass ratio RNA Y(RKH)2R RNA g Peptid g N/P 1.00 10000.00 1.00 19.67 27.78 1.00 5000.00 1.00 9.83 13.89 1.00 2500.00 1.00 4.92 6.94 1.00 1000.00 1.00 1.97 2.78 1.00 500.00 1.00 0.98 1.39 1.00 100.00 1.00 0.20 0.28 1.00 10.00 1.00 0.02 0.03

Example 4Nona-Arginine((Arg).SUB.9.)-Mediated Transfection and Expression of Stabilized Luciferase mRNA According to SEQ ID NO: 35 or 36 (Luc-RNActive) in HeLa-Cells

[0302] Hela-cells (15010.sup.3/well) were seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out. For transfection (40 l) 50 l of the RNA/(peptide)-solution as disclosed in Example 3 were mixed with 250 l serum free medium and added to the cells (final RNA concentration: 13 g/ml). Prior to addition of the transfection solution the HeLa-cells were washed gently and carefully 2 times with 1 ml Optimen (Invitrogen) per well. Then, the transfection solution (300 l per well) was added to the cells and the cells were incubated for 4 h at 37 C. Subsequently 300 l RPMI-medium (Camprex) containing 10% FCS was added per well and the cells were incubated for additional 20 h at 37 C. The transfection solution was sucked off 24 h after transfection and the cells were lysed in 300 l lysis buffer (25 mM Tris-PO4, 2 mM EDTA, 10% glycerol, 1% Triton-X 100, 2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO.sub.4, 5 mM ATP, 62.5 M luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)). The results of these experiments are shown in FIGS. 8 and 12 to 18.

Example 5Immune Stimulation Upon Transfection of Complexes of RNA with Nona-Arginine ((Arg).SUB.9.) or Poly-L-Arginine (Comparative Example)

a) Transfection Experiments

[0303] HPBMC cells from peripheral blood of healthy donors were isolated using a Ficoll gradient and washed subsequently with 1PBS (phophate-buffered saline). The cells were then seeded on 96-well microtiter plates (200103/well). The hPBMC cells were incubated for 24 h, as described under Example 4, supra, with 10 l of the RNA/peptide complex (RNA final concentration: 6 g/ml; the same amounts of RNA were used) in X-VIVO 15 Medium (BioWhittaker) (final RNA Concentration: 10 g/ml). The immunostimulatory effect upon the hPBMC cells was measured by detecting the cytokine production (Interleukin-6 and Tumor necrose factor alpha). Therefore, ELISA microtiter plates (Nunc Maxisorb) were incubated over night (o/n) with binding buffer (0.02% NaN3, 15 mM Na2CO3, 15 mM NaHCO.sub.3, pH 9.7), additionally containing a specific cytokine antibody. Cells were then blocked with 1PBS, containing 1% BSA (bovine serum albumin). The cell supernatant was added and incubated for 4 h at 37 C. Subsequently, the microtiter plate was washed with 1PBS, 0.05% TWEEN-20 surfactant and then incubated with a Biotin-labelled secondary antibody (BD Pharmingen, Heidelberg, Germany). Streptavidin-coupled horseraddish peroxidase was added to the plate. Then, the plate was again washed with 1PBS, containing 0.05% TWEEN-20 surfactant, and AB TS (2,2-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added as a substrate. The amount of cytokine was determined by measuring the absorption at 405 nm (OD405) using a standard curve with recombinant Cytokines (BD Pharmingen, Heidelberg, Germany) with the Sunrise ELISA-Reader from Tecan (Crailsheim, Germany).

b) Results

[0304] i) Immunostimulatory Effect of RNA Complexed with Nona-Arginine ((Arg).sub.9) [0305] i1) HPBMC cells were incubated with RNA complexed with nona-arginine ((Arg).sub.9) for 24 h as disclosed above, wherein the mass ratio of RNA:(Arg).sub.9 was 1:1. Then, IL-6 production was measured in the cell supernatants using ELISA. As a result, HPBMC cells showed a significant IL-6 production, i.e. a significant immunostimulatory effect of RNA complexed with nona-arginine ((Arg).sub.9) (see FIG. 5). [0306] i2) HPBMC cells were incubated with RNA complexed with nona-arginine ((Arg).sub.9) for 24 h as disclosed above, wherein the mass ratio of RNA:(Arg).sub.9 was 1:1. Then, THF-alpha production was measured in the cell supernatants using ELISA. As a result, HPBMC cells showed a significant TNF-alpha production, i.e. a significant immunostimulatory effect of RNA complexed with nona-arginine ((Arg).sub.9) (see FIG. 6).

[0307] ii) Comparison of Immunostimulatory Effect of RNA Complexed with Either Nona-Arginine ((Arg).sub.9) or Poly-L-Arginine, Respectively (Comparative Example)

[0308] hPBMCs were incubated in different mass ratios (RNA:nona-arginine 1:10; 1:8; 1:5; 1:2; 1:1; 2:1, 5:1; 8:1 and 10:1) with a complex of RNA and nona-arginine ((Arg).sub.9) or poly-L-arginine, etc., respectively, for 24 h. Subsequently TNF-alpha production was measured using ELISA.

[0309] Advantagously, a significant immunostimulatory effect can be observed for mass ratios lower than 5:1 (RNA:nona-arginine) (1:10; 1:8; 1:5; 1:2; 1:1; 2:1) (see FIG. 7). When using mass ratios of RNA:nona-arginine (5:1) no significant TNFalpha production can be observed. The same applies to stimulation experiments, using nona-arginine ((Arg).sub.9) or mRNA alone (see FIG. 7, left).

[0310] Furthermore, complexation of mRNA with poly-L-arginine leads to significantly lower induction of TNF-alpha production in comparison to nona-arginine ((Arg).sub.9) (see FIG. 7, right). Additionally, it was observed that higher concentrations of poly-L-arginine appear to be toxic for cells transfected therewith, particularly when using a mass ratio of 1:2 RNA:poly-L-arginine or lower, since the cells were lysed.

Example 6Luciferase Expression Upon Transfection of Complexes of RNA with Nona-Arginine ((Arg).SUB.9.) or Poly-L-Arginine, Respectively, in HeLa Cells (Comparative Example)

[0311] a) Luciferase expression upon transfection of complexes of RNA with nona-arginine ((Arg).sub.9) in HeLa cells. HeLa-Cells were transfected with RNActive encoding luciferase, which has been complexed with different ratios of nona-arginine or Poly-L-Arginine, respectively. 24h later luciferase-activity was measured. Apparently, a mass ration of less than 2:1 (RNA:nona-arginine) appears to be advantageous (see FIG. 8). [0312] b) In comparison, complexation with (high molecular mass) poly-L-arginine does not increase luciferase-activity at a significant level. Thus, (high molecular mass) poly-L-arginine does not appear to be suitable for transfection of mRNA (see FIG. 8).

Example 7Luciferase Expression Upon Transfection of Complexes of RNA with Hepta-Arginine ((Arg).SUB.7.) in HeLa Cells (Comparative Example)

[0313] HeLa-Cells were transfected with RNActive encoding luciferase, which has been complexed with different ratios of hepta-arginine ((Arg).sub.7). 24h later luciferase-activity was measured. Apparently, complexation with hepta-arginine ((Arg).sub.7) does not increase luciferase-activity at a significant level. Thus, hepta-arginine ((Arg).sub.7) does not appear to be suitable for transfection of mRNA (see FIG. 9).

Example 8Immune Stimulation Upon Transfection of Complexes of RNA with Hepta-Arginine ((Arg).SUB.7.) (Comparative Example)

a) Transfection Experiments

[0314] Transfection Experiments were carried out for hepta-arginine ((Arg).sub.7) analogously to the experiments in Example 5 as shown above.

b) Results of Immunostismulatory Effect of RNA Complexed with Hepta-Arginine ((Arg).sub.7) [0315] i) HPBMC cells were incubated with RNA complexed with hepta-arginine ((Arg).sub.7) for 24 h as disclosed above, wherein the mass ratio of RNA:(Arg).sub.7 was 1:1. Then, IL-6 production was measured in the cell supernatants using ELISA. As a result, HPBMC cells showed a significant IL-6 production, i.e. a significant immunostimulatory effect of RNA complexed with hepta-arginine ((Arg).sub.7) (see FIG. 10). [0316] ii) HPBMC cells were furthermore incubated with RNA complexed with hepta-arginine ((Arg).sub.7) for 24 h as disclosed above, wherein the mass ratio of RNA:(Arg).sub.7 was 1:1. Then, THF-alpha production was measured in the cell supernatants using ELISA. As a result, HPBMC cells also showed a significant TNF-alpha production, i.e. a significant immunostimulatory effect of RNA complexed with hepta-arginine ((Arg).sub.7) (see FIG. 11).

Example 9Determination of the Effect of Histidin on the Transfection Efficiency

[0317] To determine the effect of Histidin on the transfection efficiency a transfection was carried out analogously to the transfection experiments above using peptides with different Histidine content. Therefore, 4 g stabilized luciferase mRNA according to SEQ ID NO: 36 (Luc-RNActive) were mixed in molar ratios with the respectively peptide (according to formula I), particularly R9, R9H3 or H3R9H3, thereby forming a complex. Afterwards the resulting solution was adjusted with water to a final volume of 50 l and incubated for 30 minutes at room temperature. The used ratios are in each experiment 1:10000, 1:5000 and 1:1000. HeLa-cells (15010.sup.3/well) were then seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out. For transfection 50 l of the RNA/(peptide)-solution were mixed with 250 l serum free medium and added to the cells (final RNA concentration: 13 g/ml). Prior to addition of the transfection solution the HeLa-cells were washed gently and carefully 2 times with 1 ml Optimen (Invitrogen) per well. Then, the transfection solution (300 l per well) was added to the cells and the cells were incubated for 4 h at 37 C. Subsequently 300 l RPMI-medium (Camprex) containing 10% FCS was added per well and the cells were incubated for additional 20 h at 37 C. The transfection solution was sucked off 24 h after transfection and the cells were lysed in 300 l lysis buffer (25 mM Tris-PO4, 2 mM EDTA, 10% glycerol, 1% Triton-X 100, 2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO4, 5 mM ATP, 62.5 M luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).

[0318] The results are shown in FIG. 19. As can be seen, a stretch of 3 histidines at one terminal end already increases the transfection efficacy of the complexed RNA, wherein a stretch of 3 histidines at both terminal ends significantly increases the transfection efficacy of the complexed RNA.

Example 10Determination of the Effect of Neutral Amino Acids on the Transfection Efficiency

[0319] To determine the effect of neutral amino acids on the transfection efficiency a further transfection experiment was carried out analogously to the transfection experiments above in Example 9 using the peptide H3R9CCS. The results of this additional experiment are shown in FIG. 20.

Example 11Immunostimulation Using R9H3 in hPBMCs

[0320] The effect of R9H3 on immunostimulation was tested in hPBMCs. Therefore, a complex of R9H3 and RNA as shown above in Example 3 was prepared. Furthermore, HPBMC cells from peripheral blood of healthy donors were isolated using a Ficoll gradient and washed subsequently with 1PBS (phophate-buffered saline). The cells were then seeded on 96-well microtiter plates (20010.sup.3/well). The hPBMC cells were incubated for 24 h, as described under Example 4, supra, with 10 l of the RNA/peptide complex (RNA final concentration: 6 g/ml; the same amounts of RNA were used) in X-VIVO 15 Medium (BioWhittaker). The immunostimulatory effect upon the hPBMC cells was measured by detecting the cytokine production (Interleukin-6 and Tumor necrose factor alpha). Therefore, ELISA microtiter plates (Nunc Maxisorb) were incubated over night (o/n) with binding buffer (0.02% NaN3, 15 mM Na2CO3, 15 mM NaHCO.sub.3, pH 9.7), additionally containing a specific cytokine antibody. Cells were then blocked with 1PBS, containing 1% BSA (bovine serum albumin). The cell supernatant was added and incubated for 4 h at 37 C. Subsequently, the microtiter plate was washed with 1PBS, 0.05% TWEEN-20 surfactant and then incubated with a Biotin-labelled secondary antibody (BD Pharmingen, Heidelberg, Germany). Streptavidin-coupled horseraddish peroxidase was added to the plate. Then, the plate was again washed with 1PBS, containing 0.05% TWEEN-20 surfactant, and AB TS (2,2-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added as a substrate. The amount of cytokine was determined by measuring the absorption at 405 nm (OD405) using a standard curve with recombinant Cytokines (BD Pharmingen, Heidelberg, Germany) with the Sunrise ELISA-Reader from Tecan (Crailsheim, Germany). The results are seen in FIGS. 21 and 22. As can be seen, a significant immunostimulation was exhibited at a ratio of 1:5000 RNA:R9H3.