COMPLEXATION OF NUCLEIC ACIDS WITH DISULFIDE-CROSSLINKED CATIONIC COMPONENTS FOR TRANSFECTION AND IMMUNOSTIMULATION

Abstract

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid (molecule) and disulfide-crosslinked cationic components as a (preferably non-toxic and non-immunogenic) polymeric carrier. The inventive polymeric carrier cargo complex allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an (innate and/or adaptive) immune response, preferably dependent on the nucleic acid to be transported as a cargo. The present invention also provides, pharmaceutical compositions, particularly vaccines and adjuvants, comprising the inventive polymeric carrier cargo complex and optionally an antigen, as well as the use of such the inventive polymeric carrier cargo complex and optionally an antigen for transfecting a cell, a tissue or an organism, for (gene-)therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned above. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and/or the inventive pharmaceutical composition, adjuvant or vaccine in one or more parts of the kit.

Claims

1. A polymeric carrier cargo complex, comprising: a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and as a cargo at least one nucleic acid molecule.

2. The polymeric carrier cargo complex according to claim 1, wherein the at least one nucleic acid molecule is an RNA.

3. The polymeric carrier cargo complex according to claim 2, wherein the at least on nucleic acid molecule is an immunostimulatory nucleic acid.

4. The polymeric carrier cargo complex according to claim 2, wherein the at least on nucleic acid molecule is an immunostimulatory RNA (isRNA) or an mRNA.

5. A polymeric carrier cargo complex according to claim 2, wherein the nitrogen/phosphate (N/P) ratio of the cationic components to the at least one nucleic acid molecule is in the range of 0.1-20, or in the range of 0.1-5, or in the range of 0.1-1.

6. A polymeric carrier cargo complex according to claim 1, wherein the polymeric carrier further comprises functional peptides or proteins.

7. A polymeric carrier cargo complex according to claim 6, wherein the functional peptides or proteins are peptide or protein antigens or antigen epitopes.

8. A polymeric carrier cargo complex according to claim 2, wherein the polymeric carrier further comprises a ligand.

9. A polymeric carrier cargo complex according to claim 8, wherein the ligand is mannose.

10. The polymeric carrier cargo complex according to claim 2, wherein the cationic components are cationic peptides.

11. The polymeric carrier cargo complex according to claim 10, wherein the cationic peptides are selected from peptides according to formula (I)
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x, wherein l+m+n+o+x=3-100, and l, m, n or o=independently of each other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90 and 91-100, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the cationic peptide; and Xaa is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x=any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and 91-100 provided that the overall content of Xaa does not exceed 90% of all amino acids of the cationic peptide, or are selected from peptides according to subformula (Ia)
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa′).sub.x(Cys).sub.y} or from peptides according to subformula (Ib)
Cys.sub.1{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cys.sub.2 wherein (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; and x are as defined above; Xaa′ is any amino acid selected from native or non-native amino acids except of Arg, Lys, His, Orn; or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and 91-100 provided that the overall content of Arg (Arginine), Lys (Lys.sub.1 ne), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide and wherein Cys.sub.1 and Cys.sub.2 are Cysteines proximal to, or terminal to (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x.

12. The polymeric carrier cargo complex according to claim 1, wherein the disulfide-bonds are formed by cysteine residues contained in the cationic peptides.

13. A polymeric carrier cargo complex according to claim 12, wherein the cysteine residue is located at the terminal ends of the cationic peptides.

14. A method of stimulating an immune response in a subject comprising administering to the subject a polymeric carrier cargo complex according to claim 1.

15. A vaccine comprising a polymeric carrier cargo complex as defined according to claim 1 and an antigen or RNA encoding an antigen.

Description

FIGURES

[0328] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0329] FIG. 1: shows the luciferase expression in HepG2 cells after transfection with polymeric carrier cargo complexes comprising 5 μg mRNA coding for luciferase (R1180). CR.sub.12C/R1180 indicates the inventive polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C (Cys-Arg.sub.12-Cys; SEQ ID NO: 6) and the mRNA R1180 as nucleic acid cargo. R.sub.12/R1180 indicates a non-inventive carrier cargo complex formed by the non-polymerizing cationic peptide R12 (Arg.sub.12) and the mRNA R1180 as nucleic acid cargo for the purpose of comparison. [0330] The complexes contain cationic peptide and nucleic acid in a mass ratio at 1:2 (w/w) or 2:1 (w/w) as indicated. [0331] R1180 represents cells which were transfected with uncomplexed RNA. [0332] Buffer represents the negative control for non-transfected cells. [0333] After 24h the level of luciferase was quantified in the lysates by chemoluminescence measurement of luciferin oxidation. [0334] Data points indicate individual biological replicates. [0335] The results firstly show that the inventive polymeric carrier cargo complexes (CR.sub.12C/R1180) induce higher levels of luciferase expression than the non-inventive carrier cargo complexes (R.sub.12/R1180) with the non-polymerized peptide R.sub.12 (SEQ ID NO: 397). And secondly it could be shown that N/P ratios higher than 1 are advantageous for in vitro transfection.

[0336] FIG. 2: shows the (in vitro) luciferase expression in HepG2 and B 16F10 cells after transfection with polymeric carrier cargo complexes comprising 5 μg mRNA coding for luciferase (R1180). CR.sub.12C/R1180 indicates the inventive polymeric carrier cargo complex formed by the disulfide cross-linked cationic peptide CR.sub.12C (Cys-Arg.sub.12-Cys; SEQ ID NO: 6) and the mRNA R1180 as nucleic acid cargo. R12/R1180 indicates a non-inventive carrier cargo complex formed by the non-polymerizing cationic peptide R.sub.12(Arg.sub.12) and the mRNA R1180 as nucleic acid cargo for the purpose of comparison. CR.sub.9C/R1180 indicates the inventive polymeric carrier cargo complex formed by the polymerized cationic peptide CR.sub.9C (Cys-Arg.sub.9-Cys; SEQ ID NO: 3) and the mRNA R1180 as nucleic acid cargo. [0337] The complexes contain cationic peptide and nucleic acid in a mass ratio at 2:1 (w/w). [0338] R1180 represents cells which were transfected with uncomplexed RNA. [0339] Buffer represents the negative control for non-transfected cells. [0340] After 24h the level of luciferase was quantified in the lysates by chemoluminescence measurement of luciferin oxidation. [0341] Data points indicate individual biological replicates. [0342] The results show that the inventive polymeric carrier cargo complexes (CR.sub.12C/R1180 and CR.sub.9C/R1180) induce higher levels of luciferase expression than the non-inventive carrier cargo complexes (R.sub.12/R1180 and R.sub.9/1180) with the non-polymerized peptides R9 (SEQ ID NO: 394) and R.sub.12(SEQ ID NO: 397).

[0343] FIG. 3: shows the in vivo expression of luciferase after intradermal injection in female BALB/c mice of 5 μg mRNA coding for luciferase (R1180). CR.sub.12C/R1180 indicates the inventive polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C (Cys-Arg.sub.12-Cys; SEQ ID NO: 6) and the mRNA R1180 as nucleic acid cargo. R.sub.12/R1180 indicates a non-inventive carrier cargo complex formed by the non-polymerizing cationic peptide R12 (Arg.sub.12; SEQ ID NO: 397) and the mRNA R1180 as nucleic acid cargo for the purpose of comparison.

[0344] The complexes contain cationic peptide and nucleic acid in a ratio of 1:2 (w/w) or 2:1 as indicated.

[0345] After 24h the level of luciferase was quantified in the tissue lysates by a chemoluminescence assay. [0346] The results firstly show that the inventive polymeric carrier cargo complexes (CR.sub.12C/R1180) induce higher levels of luciferase expression than the non-inventive carrier cargo complexes (R.sub.12/R1180) with the non-polymerized peptide R.sub.12 (SEQ ID NO: 397; in fact no luciferase expression in the sample with the non-polymerized peptide R.sub.12could be detected). And secondly it could be shown that N/P ratios below 1 are advantageous for in vivo transfection.

[0347] FIG. 4: shows the raw correlation curve of inventive polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptides CR.sub.12C (SEQ ID NO: 6) and CRTC (SEQ ID NO: 1) as carrier after lyophilisation compared to non-inventive complexes with non-polymerizing cationic peptides as carrier (R.sub.12(SEQ ID NO: 397) and R7 (SEQ ID NO: 392)) by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK). The hydrodynamic diameters were measured with fresh prepared complexes and with reconstituted complexes after lyophilisation The mass ratio of peptide:RNA was 1:2. As result it can be shown that the inventive polymeric carrier cargo complexes comprising cystein-containing peptides as cationic components which leads to a polymerization of the polymeric carrier by disulfide bonds do not change in size in contrast to the complexes formed by non-polymerizing peptides which increase in size and therefore are not stable during the lyophilization step. Therefore complexes with polymerized peptides as polymeric carriers show advantageous properties for lyophilization.

[0348] FIG. 5: shows the Zeta-potential of inventive polymeric carrier cargo complexes formed by the disulfide-cross-linked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the R722 as nucleic acid cargo at different w/w ratios according to the invention. As can be seen, the zeta potential changes from positive to negative when the w/w ratio is changed from excess peptide to a 1:1 ratio (peptide/RNA).

[0349] FIG. 6A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the CpG 2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/CpG 2216) according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase of hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0350] FIG. 6B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the CpG 2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/CpG 2216) according to the invention. As can be seen, the inventive polymeric carrier cargo complexes do not lead to an increase in hTNFα cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0351] FIG. 7A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the mRNA R491 coding for luciferase as nucleic acid cargo in a mass ratio of 1:2 (w/w) (CR.sub.12C/R491) according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase of hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0352] FIG. 7B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the mRNA R491 coding for luciferase as nucleic acid cargo in a mass ratio of 1:2 (w/w) (CR.sub.12C/R491) according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase of hTNFα cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0353] FIG. 8A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and a short GU rich RNA oligonucleotide (short GU rich) as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/short GU rich) according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase of hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0354] FIG. 8B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and a short GU rich RNA oligonucleotide (short GU rich) as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/short GU rich) according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase of hTNFα cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0355] FIG. 9A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.7C (SEQ ID NO: 6) and the long non-coding GU-rich isRNA R722 as nucleic acid cargo according to the invention. As can be seen, the inventive polymeric carrier cargo complexes (CR.sub.7C/R722) lead to an increase of hIFNa cytokine release in hPBMCs compared to non-inventive carrier cargo complexes (R.sub.7/R722) formed by the non-polymerized peptide R.sub.7 (SEQ ID NO: 392).

[0356] FIG. 9B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.7C (SEQ ID NO: 6) and the long non-coding GU-rich isRNA R722 as nucleic acid cargo according to the invention. As can be seen, the inventive polymeric carrier cargo complexes (CR.sub.7C/R722) only leads to a weak increase of hTNFα cytokine release in hPBMCs compared to non-inventive carrier cargo complexes (R.sub.7/R722) formed by the non-polymerized peptide R.sub.7 (SEQ ID NO: 392).

[0357] FIG. 10A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.9C (SEQ ID NO: 3) and the long non-coding GU-rich isRNA R722 as nucleic acid cargo according to the invention. As can be seen, the inventive polymeric carrier cargo complexes (CR.sub.9C/R722) lead to an increase of hIFNa cytokine release in hPBMCs compared to non-inventive carrier cargo complexes (R.sub.9/R722) formed by the non-polymerized peptide R.sub.9 (SEQ ID NO: 394).

[0358] FIG. 10B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.9C (SEQ ID NO: 3) and the long non-coding GU-rich isRNA R722 as nucleic acid cargo according to the invention. As can be seen, the inventive polymeric carrier cargo complexes (CR.sub.9C/R722) do not lead to an increase of hTNFα cytokine release in hPBMCs compared to non-inventive carrier cargo complexes (R.sub.9/R722) formed by the non-polymerized peptide R.sub.9 (SEQ ID NO: 394).

[0359] FIG. 11A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the isRNA R722 as nucleic acid cargo at different w/w ratios according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase in hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0360] FIG. 11B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) and the isRNA R722 as nucleic acid cargo at different w/w ratios according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase in hTNFα cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0361] FIG. 12A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier complexes formed by the cationic peptides CH.sub.6R.sub.4H.sub.6C (SEQ ID NO: 388), CH.sub.3R.sub.4H.sub.3C (SEQ ID NO: 390) and CHK.sub.7HC (SEQ ID NO: 389) and the isRNA R722 as nucleic acid cargo at different N/P ratios according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase in hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

[0362] FIG. 12B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier complexes formed by the disulfide-crosslinked cationic peptides CH.sub.6R.sub.4H.sub.6C (SEQ ID NO: 388), CH.sub.3R.sub.4H.sub.3C (SEQ ID NO: 390) and CHK.sub.7HC (SEQ ID NO: 389) and the isRNA R722 as nucleic acid cargo at different N/P ratios according to the invention. As can be seen, the inventive polymeric carrier cargo complexes lead to an increase in hTNFα cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone. Particularly inventive polymeric cargo complexes with an N/P ratio greater or equal 1 result in TNFalpha secretion.

[0363] FIG. 13: shows the (in vivo) effect of the addition of the inventive polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) as carrier and the isRNA R722 as nucleic acid cargo to the protein vaccine Ovalbumine (OVA protein) for the use as an adjuvant in tumour challenge experiments. As can be seen, the inventive polymeric carrier cargo complex extremely decelaterates the tumour growth compared to the protein vaccine alone, which has no effect on tumor growth in comparison to the buffer control.

[0364] FIG. 14: shows the (in vivo) effect of the addition of the inventive polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) as carrier and the isRNA R722 as nucleic acid cargo to the protein vaccine Ovalbumine (OVA protein) for the use as an adjuvant on the induction of Ovalbumine-specific IgG2a antibodies. As can be seen, the inventive polymeric carrier cargo complex strongly increases the B-cell response, which proofs the beneficial adjuvant properties of the inventive polymeric carrier cargo complexes, particularly in regard to the induction of a Th1-shifted immune response.

[0365] FIG. 15: shows the (in vivo) effect of the addition of the inventive polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C (SEQ ID NO: 6) as carrier and the isRNA R722 as nucleic acid cargo to the protein vaccine Ovalbumine (OVA protein) or the Ovalbumine-specific peptide vaccine SIINFEKL (SEQ ID NO: 535) for the use as an adjuvant on the induction of Ovalbumine-specific cytotoxic T cells. As can be seen, the inventive polymeric carrier cargo complex strongly increases the induction of Ovalbumin-specific cytotoxic T cells compared to the vaccination with protein or peptide alone, which further proofs the beneficial adjuvant properties of the inventive polymeric carrier cargo complex, particularly in regard to the induction of a Th1-shifted immune response.

EXAMPLES

[0366] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0367] 1. Reagents:

[0368] Cationic Peptides as Cationic Component:

TABLE-US-00005 R.sub.7: (SEQ ID NO: 392) Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.7) CR.sub.7C: (SEQ ID NO: 1) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (CysArg.sub.7Cys) R.sub.9: (SEQ ID NO: 394) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.9) R.sub.12: (SEQ ID NO: 397) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.12) CR.sub.9C: (SEQ ID NO: 3) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (Cys- Arg.sub.9-Cys) CR.sub.12C: (SEQ ID NO: 6) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Cys-Arg.sub.12-Cys)

[0369] Nucleic Acids as Cargo: [0370] R1180: mRNA coding for luciferase (SEQ ID NO. 384) [0371] R722: long non-coding is GU-rich RNA (SEQ ID NO. 385) [0372] R491: mRNA coding for luciferase (SEQ ID NO. 386) [0373] CpG 2216: CpG oligonucleotide (SEQ ID NO. 387) [0374] Short GU rich: GU-rich RNA oligonucleotide (SEQ ID NO. 369)

[0375] 2. Preparation of Nucleic Acid Sequences:

[0376] For the present examples nucleic acid sequences as indicated in example 1 were prepared and used for formation of the inventive polymerized polymeric carrier cargo complexes or for non-polymerized carrier cargo complexes for comparison. These polymeric carrier cargo complexes were used for in vitro and in vivo transaction, for in vitro immunostimulation and for particle characterizations.

[0377] According to a first preparation, the DNA sequences, coding for the corresponding RNA sequences R1180, R722 and R491 sequences were prepared. The sequences of the corresponding RNAs are shown in the sequence listing (SEQ ID NOs: 384, 385 and 386).

[0378] The short GU rich sequences and the CpG 2216 oligonucleotides were prepared by automatic solid-phase synthesis by means of phosphoramidite chemistry. The sequences are shown in the sequence listing (SEQ ID NOs: 387 and 369).

[0379] 2. In Vitro Transcription:

[0380] The respective DNA plasmids prepared according to Example 2 for R1180, R722 and R491 were transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Germany) following the manufactures instructions. Subsequently the mRNA was purified using PureMessenger® (CureVac, Tubingen, Germany).

[0381] 3. Synthesis of Polymeric Carrier Cargo Complexes:

[0382] The nucleic acid sequences defined above in Example 1 were mixed with the cationic components as defined in Example 1. Therefore, the indicated amount of nucleic acid sequence was mixed with the respective cationic component in mass ratios as indicated, thereby forming a complex. If polymerizing cationic components were used according to the present invention polymerization of the cationic components took place simultaneously to complexation of the nucleic acid cargo. Afterwards the resulting solution was adjusted with water to a final volume of 50 μl and incubated for 30 min at room temperature. The different ratios of cationic component/nucleic acid used in the experiments are shown in Table 1.

TABLE-US-00006 TABLE 1 Sample (cationic peptide/nucleic acid) Mass ratio N/P ratio Molar ratio CR.sub.12C 1:2 0.9  44:1 (SEQ ID NO: 6)/R1180 CR.sub.12C 2:1 3.6 185:1 (SEQ ID NO: 6)/R1180 R.sub.12 1:2 0.7  48:1 (SEQ ID NO: 397)/R1180 R.sub.12 2:1 2.5 146:1 (SEQ ID NO: 397)/R1180 CR.sub.9C 2:1 0.9  55:1 (SEQ ID NO: 3)/R1180 R.sub.9 2:1 1.1  65:1 (SEQ ID NO: 394)/R1180 CR.sub.7C (SEQ ID NO: 1) 1:2 0.8  70:1 R.sub.7 (SEQ ID NO: 392) 1:2 1.0  85:1 CR.sub.12C 1:2, 5 4.9  8:1 (SEQ ID NO: 6)/CpG CR.sub.12C 1:2 0.9 150:1 (SEQ ID NO: 6)/R491 CR.sub.12C 1:2, 5 4.9  8:1 (SEQ ID NO: 6)/ short GU-rich CR.sub.12C 5:1 9.6 444:1 (SEQ ID NO: 6)/R722 CR.sub.12C 4:1 7.6 355:1 (SEQ ID NO: 6)/R722 CR.sub.12C 3:1 5.7 266:1 (SEQ ID NO: 6)/R722 CR.sub.12C 2:1 3.8 177:1 (SEQ ID NO: 6)/R722 CR.sub.12C 1:1 1.9  88:1 (SEQ ID NO: 6)/R722 CR.sub.12C 1:2 0.9  44:1 (SEQ ID NO: 6)/R722 CR.sub.12C 1:3 0.6  29:1 (SEQ ID NO: 6)/R722 CR.sub.12C 1:4 0.5  22:1 (SEQ ID NO: 6)/R722 CR.sub.12C 1:5 0.4  17:1 (SEQ ID NO: 6)/R722 N/P ratio = is a measure of the ionic charge of the cationic component of the polymeric carrier or of the polymeric carrier as such. In the case that the cationic properties of the cationic component are provided by nitrogen atoms the N/P ratio is the ratio of basic nitrogen atoms to phosphate residues, considering that nitrogen atoms confer to positive charges and phosphate of the phosphate backbone of the nucleic acid confers to the negative charge. N/P is preferably calculated by the following formula: [00001]$N/P=\frac{\mathrm{pmol}\left[\mathrm{RNA}\right]*\mathrm{ratio}*\mathrm{cationic}\mathrm{AS}}{\mathrm{.Math.g}\mathrm{RNA}*3*1000}$As an example the RNA R722 according to SEQ ID NO: 385 was applied, which has a molecular weight of 186 kDa. Therefore 1 μg R722 RNA confers to 5.38 pmol RNA.

[0383] 4. Transfection of HepG2 and B16F10 Cells:

[0384] Polymeric carrier cargo complexes containing 5 μg mRNA coding for luciferase (R1180) were prepared as indicated in Example 3. HepG2 or B16F10 cells (150×10.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 50 μl of the polymeric carrier cargo complex solution were mixed with 250 μl serum free medium and added to the cells (final RNA concentration: 13 μg/ml). Prior to addition of the serum free transfection solution the cells were washed gently and carefully 2 times with 1 ml OPTI-MEM (Invitrogen) per well. Then, the transfection solution (300 μl per well) was added to the cells and the cells were incubated for 4 h at 37° C. Subsequently 100 μl 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 μl lysis buffer (25 mM Tris-PO.sub.4, 2 mM EDTA, 10% glycerol, 1% Triton-X 100, 2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO.sub.4, 5 mM ATP, 62.5 μM luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).

[0385] 5. Expression of Luciferase In Vivo:

[0386] Polymeric carrier cargo complexes containing 5 μg mRNA coding for luciferase (R1180) were prepared as indicated in Example 3. Afterwards the resulting solution was adjusted with Ringer Lactate solution to a final volume of 100 μl and incubated for 30 minutes at room temperature, yielding a solution with a 0.1 g/l concentration of polymeric carrier cargo complexes. 100 μl of this solution was administrated intradermally (in the back) of 7 week old BALB/c mice. After 24h the mice were sacrificed and the samples (skin from the back) were collected, frozen at −78° C. and lysed for 3 Minutes at full speed in a tissue lyser (Qiagen, Hilden, Germany). Afterwards 600 μl of lysis buffer were added and the resulting solutions were subjected another 6 minutes at full speed in the tissue lyser. After 10 minutes centrifugation at 13500 rpm at 4° C. the supernatants were mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO.sub.4, 5 mM ATP, 62.5 μM luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).

[0387] 6. Cytokine Stimulation in hPBMCs:

[0388] HPBMC cells from peripheral blood of healthy donors were isolated using a Ficoll gradient and washed subsequently with 1(PBS (phophate-buffered saline). The cells were then seeded on 96-well microtiter plates (200×10.sup.3/well). The hPBMC cells were incubated for 24 h with 10 μl of the inventive polymeric carrier cargo complex from Example 3 containing the indicated amount of nucleic acid in X-VIVO 15 Medium (BioWhittaker). The immunostimulatory effect was measured by detecting the cytokine production of the hPBMCs (Tumour necrose factor alpha and Interferon alpha). Therefore, ELISA microtiter plates (Nunc MAXISORP) 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 1×PBS, 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 1×PBS, containing 0.05% TWEEN-20 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 1×PBS, containing 0.05% TWEEN-20 and ABTS (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 (OD 405) using a standard curve with recombinant cytokines (BD Pharmingen, Heidelberg, Germany) with the SUNRISE ELISA-Reader from Tecan (Crailsheim, Germany).

[0389] 7. Zetapotential Measurements:

[0390] The Zeta potential of the polymeric carrier cargo complexes was evaluated by the laser Doppler electrophoresis method using a Zetasizer Nano (Malvern Instruments, Malvern, UK). The measurement was performed at 25° C. and a scattering angle of 173° was used.

[0391] 8. Stability of Complexes after Lyophilization

[0392] The hydrodynamic diameters of polymeric carrier cargo complexes as prepared above were measured by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK) according to the manufacturer's instructions. The measurements were performed at 25° C. in buffer analysed by a cumulant method to obtain the hydrodynamic diameters and polydispersity indices of the polymeric carrier cargo complexes. Polymeric carrier cargo complexes were formed as indicated in Example 3 and the hydrodynamic diameters were measured with fresh prepared complexes and with reconstituted complexes after lyophilization.

[0393] 9. Immunization Experiments:

[0394] For immunization the vaccines Ovalbumine protein (OVA) (5 μg) or Ovalbumin-specific peptide SIINFEKL (SEQ ID NO: 535; 50 μg) were combined with the inventive polymeric cargo complexes R722/CR.sub.12C (SEQ ID NO: 6) (in a ratio of 2:1 w/w) (30 μg R722/15 μg CR.sub.12C (SEQ ID NO: 6)). as adjuvant and injected intradermally into female C57BL/6 mice (7 mice per group for tumour challenge and 5 mice per group for detection of an immune response). The vaccination was repeated 2 times in 2 weeks. For comparison mice were injected without the inventive polymeric cargo complexes.

[0395] 10. Detection of an Antigen-Specific Immune Response (B-Cell Immune Response):

[0396] Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting antigen specific antibodies. Therefore, blood samples were taken from vaccinated mice 5 days after the last vaccination and sera were prepared. MaxiSorb plates (Nalgene Nunc International) were coated with Gallus gallus ovalbumine protein. After blocking with 1×PBS containing 0.05% TWEEN-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate was incubated with Horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured.

[0397] 11. Detection of an Antigen Specific Cellular Immune Response by ELISPOT:

[0398] days after the last vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INF□ (BD Pharmingen, Heidelberg, Germany). The next day 1×10.sup.6 cells/well were added and re-stimulated with 1 μg/well of relevant peptide (SIINFEKL of ovalbumine; SEQ ID NO; 535); irrelevant peptide (Connexin=control peptide) or buffer without peptide. Afterwards the cells are incubated for 24h at 37° C. The next day the plates were washed 3 times with PBS, once with water and once with PBS/0.05% TWEEN-20 and afterwards incubated with a biotin-coupled secondary antibody for 11-24h at 4° C. Then the plates were washed with PBS/0.05% TWEEN-20 and incubated for 2 h at room temperature with alkaline phosphatase coupled to streptavidin in blocking buffer. After washing with PBS/0.05% TWEEN-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The reaction was then stopped by washing the plates with water. The dried plates were then read out by an ELISPOT plate reader. For visualization of the spot levels the numbers were corrected by background subtraction.

[0399] 12. Tumour Challenge:

[0400] One week after the last vaccination 1×10.sup.6 E.G7-OVA cells (tumour cells which stably express ovalbumine) were implanted subcutaneously in the vaccinated mice. Tumour growth was monitored by measuring the tumour size in 3 dimensions using a calliper.