Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids

Abstract

The present invention is directed to an inventive polymeric carrier molecule according to generic formula (I) and variations thereof, which allows for efficient transfection of nucleic acids into cells in vivo and in vitro, a polymeric carrier cargo complex formed by a nucleic acid and the inventive polymeric carrier molecule, but also to methods of preparation of this inventive polymeric carrier molecule and of the inventive polymeric carrier cargo complex. The present invention also provides methods of application and use of this inventive polymeric carrier molecule and the inventive polymeric carrier cargo complex as a medicament, for the treatment of various diseases, and in the preparation of a pharmaceutical composition for the treatment of such diseases.

Claims

1. A pharmaceutical composition comprising: (A) a polymeric carrier molecule according to formula (I):
L-P.sup.1S[SP.sup.2S].sub.nSP.sup.3-L wherein, P.sup.1 and P.sup.3 are different or identical to each other and represent a linear or branched hydrophilic polyethylene glycol (PEG) polymer chain, wherein the hydrophilic PEG polymer chain exhibits a molecular weight of 1 kDa to 100 kDa; P.sup.2 is a cationic or polycationic polypeptide, having a length of 3 to 100 amino acids, and comprising at least 2 cysteine residues; SS is a (reversible) disulfide bond, wherein one of the sulfur positions of each of the disulfide bonds is provided by the at least 2 cysteine residues of the polypeptide of P.sup.2; L is an optional ligand, which may be present or not, and may be selected independent from the other from a localization signal or sequence, an antibody, a cell penetrating peptide, a ligand of a receptor, small molecules, carbohydrates, or inhibitors or antagonists of receptors; and n is an integer, selected from a range of 1 to 50; and (B) an adjuvant component.

2. The pharmaceutical composition of claim 1, wherein the adjuvant component comprises cytokine, lipopolysaccharide (LPS) or toll-like receptor (TLR) ligand.

3. The pharmaceutical composition of claim 2, wherein the adjuvant component comprises a ligand of human TLR 1-10.

4. The pharmaceutical composition of claim 2, wherein the adjuvant component comprises LPS.

5. The pharmaceutical composition of claim 2, wherein the adjuvant component comprises granulocyte macrophage colony-stimulating factor (GM-CSF).

6. The pharmaceutical composition of claim 1, wherein the adjuvant component comprises CpG-DNA or an immunostimulatory RNA (isRNA).

7. The pharmaceutical composition of claim 1, wherein the adjuvant component comprises an immunostimulatory polypeptide.

8. The pharmaceutical composition of claim 1, wherein component P.sup.2 of the polymeric carrier is selected from a peptide comprising a cationic peptide of formula (IIb):
Cys{(Arg).sub.l(Lys).sub.m(His).sub.n(Orn).sub.o(Xaa).sub.x}Cys, wherein l+m+n+o+x=8-16, 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 10% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the oligopeptide.

9. The pharmaceutical composition of claim 1, wherein the polymeric carrier further comprises a nucleic acid molecule.

10. The pharmaceutical composition of claim 9, wherein the nucleic acid is provided in a molar ratio of 5 to 10000 of polymeric carrier molecule: nucleic acid.

11. The pharmaceutical composition of claim 9, wherein the nucleic acid is a DNA or a RNA.

12. The pharmaceutical composition of claim 9, wherein the nucleic acid is a mRNA, an siRNA, or an immunostimulatory RNA (isRNA).

13. The pharmaceutical composition of claim 9, wherein the nucleic acid is the adjuvant component of said composition and comprises an isRNA or CpG-DNA molecule.

14. The pharmaceutical composition of claim 9, wherein the nucleic acid encodes an antigen.

15. The pharmaceutical composition of claim 14, wherein the nucleic acid encodes a tumor antigen, or a pathogenic antigen.

16. The pharmaceutical composition of claim 9, wherein the nucleic acid encodes an immunostimulatory polypeptide.

17. The pharmaceutical composition of claim 1, wherein component P.sup.2 comprises a cationic peptide of formula (IIb): CysHis6Arg4His6Cys (SEQ ID NO: 83).

18. The pharmaceutical composition of claim 1, wherein one of the sulfur positions of each of the disulfide bonds of formula (I) is provided by a cysteine residue at the N- or C-terminus of the polypeptide of P.sup.2.

19. The pharmaceutical composition of claim 1, further comprising a lipid component.

20. The pharmaceutical composition of claim 19, wherein the lipid component comprises a cationic lipid.

21. The pharmaceutical composition of claim 1, wherein component P.sup.2 is a cationic or polycationic polypeptide selected from a cell penetrating peptide (CPP), an HIV-binding peptide, a member of the penetratin family, or a protein transduction domain (PTD).

22. The pharmaceutical composition of claim 1, wherein L is (i) a localization signal or sequence selected from RGD, Transferrin, a signal peptide or signal sequence, or a nuclear localization signal or sequence (NLS), (ii) a small molecule selected from folate, a synthetic ligand or a small molecule agonist, (iii) TAT, (iv) a ligand of a receptor selected from a cytokine, a hormone, or a growth factor; a carbohydrate selected from mannose or galactose; or (v) an RGD peptidomimetic analog.

23. The pharmaceutical composition of claim 15, wherein the pathogenic antigen is an animal antigen, a viral antigen, a protozoal antigen, a bacterial antigen, an allergic antigen, an autoimmune antigen, or an allergen.

24. The pharmaceutical composition of claim 21, wherein (i) the CPP is protamine, nuceloline, spermine, spermidine, an L-oligomer, an arginine-rich peptide, a chimeric CPP, or a member of the penetratin family; (ii) the HIV-binding peptide is Tat.

25. The pharmaceutical composition of claim 24, wherein (i) the member of the penetratin family is Penetratin, pAntp, pIsl, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, SAP, MAP, KALA, PpTG20, a proline-rich peptide, a Loligomere, an arginine-rich peptide, Calcitonin peptide, FGF, Lactoferrin, histone, VP22 peptide, or Herpes simplex virus VP22; (ii) the chimeric CPP is Transportan or an MGP peptide; (iii) the L-oligomer is poly-L-lysine; or (iv) the arginine-rich peptide is poly-arginine or oligoarginine.

Description

FIGURES

(1) The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

(2) FIG. 1: illustrates the products formed during the reaction by SDS PAGE gel electrophoresis under non-reducing conditions. The left part of the gel was stained according to coomassie protocol which colors the peptide content, the right part was stained with Bariumchloride/Iodine solutions which colour the PEG content. As may be easily seen only one product containing PEG and peptide is formed in the range of the intended mass.

(3) FIG. 2: shows the results of the confocal microscopy of L929 cells 5 minutes after transfection with fluorescence labelled RNA complexed with the inventive polymeric carrier PB19 in a molar ratio of 1:500. As a result, several complexes are detectable in the cells already 5 minutes after transfection indicating a good transfection rate.

(4) FIG. 3: shows the results of the confocal microscopy of L929 cells 1 hour after transfection with fluorescence labelled RNA complexed with the inventive polymeric carrier PB19 in a molar ratio of 1:500. As a result, most of the particles were taken up in the cells 1 hour after transfection showing a good transfection rate.

(5) FIG. 4: illustrates stability experiments with regard to electrostatic displacement of the bound nucleic acid from the complex. As can be seen the addition of the anionic polymer heparin can not displace the RNA from the complex because it still migrates in the gel. This indicates that the complex binding is so strong that a competitive complex partner cannot displace the RNA from the complex. The content of lanes 1-4 are indicated in the Figure from left to right (lanes 1-4).

(6) FIG. 5: depicts a gel shift assay to examine the strength of complex binding. It could be shown that the addition of the anionic polymer heparin or the reducing agent DTT alone cannot display the RNA from the complex with the polymer according to the invention (PB19). Only together they are able to displace the RNA from the complex (lane 7). This indicates that the complex binding is so strong that neither a competitive complex partner nor a reducing agent can displace the RNA from the complex.

(7) FIG. 6: shows a gel shift assay to examine the strength of complex binding. As can be seen, the addition of the anionic polymer heparin alone cannot displace the RNA from the complex with polymers according to the invention (PB22). In contrast mRNA could be readily displaced by heparin from PEI complexes.

(8) FIG. 7: depicts the results from expression experiments with of luciferase encoding mRNA according to SEQ ID NO: 369 in HeLa cells. As can be seen, formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with the PB19 polymer (molar ratio of RNA:PB19 1:1000, 1:500, 1:100) lead to expression of luciferase independently of the presence of serum containing medium. These results are unexpected because serum containing medium leads in general to a loss of transfection efficiency.

(9) FIG. 8: depicts the results from expression experiments with of luciferase encoding mRNA according to SEQ ID NO: 369 in HeLa cells. As can be seen, formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with the PB83 polymer (molar ratio of RNA:PB83 1:50) lead to significant higher expression of luciferase compared to th free peptide combined with RNA in molar ratio 1:50 (e.g. template assisted polymerization in situ Rice et. Al.), to the non-pegylated polymerization product PB83 w/o PEG in molar ratio 1:50 (e.g. RPC conform), PB83 polymerization without peptide component (e.g. dimerized PEG-SS-PEG) and a non-reversible PEGylated of PB83 derivative which was synthesized by malimide containing PEG termination of the polymerization reaction.

(10) FIG. 9A: illustrates the results from expression experiments with luciferase encoding mRNA according to SEQ ID NO: 369 in BALB/c mice after intradermal injection. As a result, formulation of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with the PB19 polymer leads to expression of luciferase in the dermis of female BALB/c mice. Other transfection reagents known in the art (PEI and Lipofectamine 2000) did not show any expression of the Luciferase protein.

(11) FIG. 9B: illustrates the results from expression experiments with luciferase encoding mRNA according to SEQ ID NO: 369 in BALB/c mice after intramuscular injection. As a result, formulation of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with the PB19 polymer leads to expression of luciferase in the m. tibialis of female BALB/c mice. Other transfection reagents known in the art (PEI and Lipofectamine 2000) did not show any expression of the Luciferase protein.

(12) FIG. 10: shows the expression of luciferase in BALB/c mice after intradermal injection of different formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with two different polymers according to the invention [PB19 and PB48]. These formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with two different polymers according to the invention [PB19 and PB48] lead to expression of luciferase in the dermis of female BALB/c mice. The polymeric carrier cargo complex formed by a peptide according to RPC CH6R4H6C (without PEGylation) and mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 in a molar ratio of 2500:1 showed no expression of luciferase after intradermal injection of the complexed mRNA.

(13) FIG. 11: shows the expression of luciferase in BALB/c mice after intradermal injection of different formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with three different polymers according to the invention [PB124, PB117 and PB83]. These formulations lead all to an expression of luciferase in the dermis of female BALB/c mice.

(14) FIG. 12: depicts the results from expression experiments with of luciferase encoding mRNA according to SEQ ID NO: 369 in CHO cells. As can be seen, formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 with different polymers according to the invention (molar ratio of RNA:PB 1:250) all lead to high levels of luciferase expression in serum containing medium. Also the advantageous effect of the hydrophilic component AA (in this case the peptide CAS.sub.3PS.sub.3AC in the polymer can easily be seen.

(15) FIG. 13: depicts the secretion of hIL-6 cytokine in hPBMCs. It could be shown that complexes consisting of the polymers according to the invention (PB19 and PB22) and mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 and cationic peptides (H3R9H3) or a combination of cationic peptides (H3R9H3) and PEGylated cationic peptides (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates). The polymers used were: E9-PEG5k: HO-PEG.sub.5000-EEEEEEEEE E9-PEG3k: HO-PEG.sub.3000-EEEEEEEEE R9-PEG3k: HO-PEG.sub.3000-RRRRRRRRR PB19: HO-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.5S-PEG.sub.5000-OH PB22: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHCS).sub.5S-PEG.sub.5000-OH

(16) FIG. 14: illustrates the secretion of hTNFa cytokine in hPBMCs. It could be shown that complexes consisting of the polymers according to the invention (PB19 and PB22) and mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 and cationic peptides (H3R9H3) or such complexes coated with PEGylated peptides (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates). The polymers used were: E9-PEG5k: HO-PEG.sub.5000-EEEEEEEEE E9-PEG3k: HO-PEG.sub.3000-EEEEEEEEE R9-PEG3k: HO-PEG.sub.3000-RRRRRRRRR PB19: HO-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.5S-PEG.sub.5000-OH PB22: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHC-5).sub.5-SPEG.sub.5000-OH

(17) FIG. 15: shows the secretion of hTFNa cytokine secretion in hPBMCs. It could be shown that complexes consisting of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 and polymers according to the invention (PB19) do not induce the secretion of hTFNa in hPBMCs in contrast to complexes consisting of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369 and state of the art transfection reagents like Lipofectamin 2000.

(18) FIG. 16: shows the mRNA sequence encoding Photinus pyralis luciferase (SEQ ID NO: 369) in the mRNA construct pCV19-Pp luc(GC)-muag-A70-C30; which exhibits a length of 1857 nucleotides. The mRNA sequence contains following sequence elements: the coding sequence encoding Photinus pyralls luciferase; stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)); 70 adenosine at the 3-terminal end (poly-A-tail); 30 cytosine at the 3-terminal end (poly-C-tail). The ORF is indicated in italic letters, muag (mutated alpha-globin-3-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line

EXAMPLES

(19) The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

(20) 1. Preparation of DNA and mRNA Constructs Encoding Pp Luciferase (Photinus pyralis)

(21) For the present examples DNA sequences, encoding Photinus pyralis luciferase, were prepared and used for subsequent in vitro transcription reactions.

(22) According to a first preparation, the DNA sequence termed pCV19-Ppluc(GC)-muag-A70-C30 sequence was prepared, which corresponds to the Photinus pyralis luciferase coding sequence. The construct was prepared by modifying the wildtype Photinus pyralis luciferase encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a stretch of 70 adenosine at the 3-terminal end (poly-A-tail) and a stretch of 30 cytosine at the 3-terminal end (poly-C-tail), leading to SEQ ID NO: 369 (see FIG. 16). The sequence of the final DNA construct had a length of 1857 nucleotides and was termed pCV19-Ppluc(GC)-muag-A70-C30. In SEQ ID NO: 369 (see FIG. 16) the sequence of the corresponding mRNA is shown.

(23) The sequence contains following sequence elements: the coding sequence encoding Photinus pyralis luciferase; stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globi n-3-UTR)); 70 adenosine at the 3-terminal end (poly-A-tail); 30 cytosine at the 3-terminal end (poly-C-tail).
2. In Vitro Transcription:

(24) The respective DNA plasmid prepared according to Example 1 was transcribed in vitro using T7-Polymerase. Subsequently the mRNA was purified using PureMessenger (CureVac, Tubingen, Germany).

(25) 3. Reagents:

(26) Peptides: The peptides used in the present experiments were as follows: PB19: HO-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.5S-PEG.sub.5000-OH (pegylated CH.sub.3R.sub.4H.sub.3C peptide polymer) PB22: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHCS).sub.5S-PEG.sub.5000-OH (pegylated CH.sub.6R.sub.4H.sub.6C peptide polymer) PB48: HO-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.3S-PEG.sub.5000-OH PB83: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHCS-).sub.7-S-PEG.sub.5000-OH PB86: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHCS-).sub.5(S-CAS3PS3ACS).sub.5S-PEG.sub.5000-OH PB117 HO-PEG.sub.5000-S(SCHKKKKKKHCS-).sub.7-S-PEG.sub.5000-OH PB124 HO-PEG.sub.2000-S(SCHHHHHHRRRRHHHHHHCS-).sub.7-S-PEG.sub.2000-OH PB83 free peptide HS(CHHHHHHRRRRHHHHHHC)SH freshly solved in water prior formulation PB83 w/o peptide PEG-SS-PEG PB83 w/o PEG HS(CHHHHHHRRRRHHHHHHC)SH PB83malPEG PEG-mal-(SCHHHHHHRRRRHHHHHHCS-).sub.x-mal-PEG H3R9H3: HHHRRRRRRRRRHHH CH6R4H6C: H(SCHHHHHHRRRRHHHHHHCS).sub.5H

(27) Further Tranfection Reagents Used are: Lipofectamine 2000 (Invitrogen) PEI 25 kDa (branched) (Aldrich)
4. Synthesis of the Inventive Polymeric Carrier:

(28) The condensation reaction was performed with the calculated amount of peptide (component P.sup.2) which is dissolved in a mixture of a buffered aqueous solution at pH 8.5 with an optional additive of 5% (v/v) Dimethylsulfoxide (DMSO) (which are mild oxidation conditions and therefore allow the establishment of an equilibrium) and stirred for 18 h at ambient temperature. Afterwards the calculated amount of a thiol group containing PEG derivative (alpha-Methoxy-omega-mercapto poly(ethylene glycol)) (component P.sup.1) (dissolved in water) is added and the resulting solution is stirred for another 18 h. Subsequent lyophilisation and purification yield the desired polymer. The ratio between PEG component P.sup.1 to peptide component P.sup.2 defines the chain length of the P.sup.2 polymer.

(29) The condensation reaction in this reaction environment is reversible, therefore the chain length of the polymer is determined by the amount of the monothiol compound which terminates the polymerisation reaction. In summary the length of the polymer chain is determined by the ratio of oligo-peptide and monothiol component. This reaction is supported by the chosen mild oxidation conditions. With more stringent oxidation conditions (30% DMSO) the generation of high molecular (long chain) polymers is induced.

(30) 4.1. 1. Step: Exemplary Polymerization Reaction:
nHSCHHHRRRHHHCSH.fwdarw.H(SCHHHRRRRHHHCS).sub.nH
4.2. 2. Step: Exemplary Stop Reaction:
H(SCHHHRRRRHHHCS).sub.nH+2PEG-SH.fwdarw.PEG-S(SCHHHRRRRHHHCS).sub.nS-PEG
4.3. Exemplary Synthesis Reaction: step 1)
5HSCHHHRRRHHHCSH.fwdarw.H(SCHHHRRRRHHHCS).sub.5H step 2)
H(SCHHHRRRRHHHCS).sub.5H+2PEG.sub.5000-SH-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.5S-PEG.sub.5000

(31) To achieve a polymer length of 5 (n=5), a molar ratio of peptide:PEG of 5:2 was used.

(32) Some variations of the synthesis reaction were done to show the the effect of the PEGchains and the effects of the reversible attachment of the PEG chains.

(33) 4.4. Synthesis Reaction for Polymeric Carriers without PEG Chains:

(34) The reaction conditions are the same as mentioned above, but the step of the addition of a sulfhydryl containing PEG derivative is not performed/skipped.

(35) 4.5. Synthesis Reaction for Irreversible Attached PEG Chains:

(36) The reaction conditions are the same as mentioned above, but instead of a sulfhydryl containing PEG derivative a maleimide containing PEG derivative is utilized. The maleimide mojety reacts rapidly with free sulfhydryl groups forming a covalent bond. Therefore the termination of the polymerization is not under the dynamic equilibria conditions as for sulfhyrdyl containing PEG derivatives but under irreversible conditions which results in a frozen polymerization pattern of high polydiversity and not the defined reaction products of the dynamic equilibria reaction.

(37) 5. Complexation of RNA:

(38) The mRNA construct defined above in Example 1 and prepared according to Example 2, were complexed for the purposes of the present invention with the polymers, preferably as defined in Example 4. Therefore, 4 g RNA coding for luciferase pCV19-Ppluc(GC)-muag-A70-C30 (Luc-RNActive) according to SEQ ID NO: 85 were mixed in molar ratios as indicated with the inventive polymeric carrier (according to formula I) or a control, 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.

(39) The different inventive polymeric carriers and the different ratios of polymeric carriers/RNA used in this experiment are shown in table 1.

(40) TABLE-US-00006 Cationic Prfix Polymer Ratio AS N/P PB19 HO-PEG.sub.5000-S-(S-CHHHRRRRHHHC-S).sub.5-S-PEG.sub.5000-OH 500 20 5.6 PB19 HO-PEG.sub.5000-S-(S-CHHHRRRRHHHC-S).sub.5-S-PEG.sub.5000-OH 250 20 2.8 PB19 HO-PEG.sub.5000-S-(S-CHHHRRRRHHHC-S).sub.5-S-PEG.sub.5000-OH 50 20 0.6 PB48 HO-PEG.sub.5000-S-(S-CHHHRRRRHHHC-S).sub.3-S-PEG.sub.5000-OH 250 12 1.67 PB76 HO-PEG.sub.5000-S-(S-CHHHHHHRRRRHHHHHHC-S).sub.10-S-PEG.sub.5000-OH 500 40 11.2 PB76 HO-PEG.sub.5000-S-(S-CHHHHHHRRRRHHHHHHC-S).sub.10-S-PEG.sub.5000-OH 250 40 5.6 PB76 HO-PEG.sub.5000-S-(S-CHHHHHHRRRRHHHHHHC-S).sub.10-S-PEG.sub.5000-OH 50 40 1.1 PB83 HO-PEG.sub.5000-S-(S-CHHHHHHRRRRHHHHHHC-S-).sub.7-S-PEG.sub.5000-OH 250 28 2.8 PB86 HO-PEG.sub.5000-S-(S-CHHHHHHRRRRHHHHHHC-S-).sub.5(S-CAS3PS3AC-S).sub.5- 250 20 2.8 S-PEG.sub.5000-OH Ratio = molar ratio of RNA:peptide cationic AS = cationic amino acids, which are positively charged at a physiological pH (i.e. not histidine (H) but e.g. arginine (R)) Whereas PB83 and PB86 have a cationic insert that is double in size compared to PB19, therefore has double as much cationic amino acid residues. PB48 has a shorter cationic insert compared to PB19, therefore has less cationic amino acid residues per molecule polymer. N/P = is the ratio of basic nitrogen atoms in the polymeric carrier to phosphate residues in the nucleic acid, considering that nitrogen atoms confer to positive charges and phosphate of the phosphate backbone of the nucleic acid confers to the negative charge. Histidine residues are counted neutral, because complex formation is done at physiological pH, therefore the imidazole residue is uncharged. N/P is calculated by the following formula: $N\text{/}P=\frac{\mathrm{pmol}\left[\mathrm{RNA}\right]*\mathrm{ratio}*\mathrm{cationic}\mathrm{AS}}{\mathrm{.Math.g}\mathrm{RNA}*3*1000}$For the calculations RNA coding for luciferase pCV19-Ppluc(GC)-muag-A70-C30 (Luc-RNActive) according to SEQ ID NO: 369 was applied, which has a molecular weight of 660 kDa. Therefore 1 g pCV19-Ppluc(GC)-muag-A70-C30 RNA according to SEQ ID NO: 369 confers to 1.67 pmol pCV19-Ppluc(GC)-muag-A70-C30 RNA according to SEQ ID NO: 369
6. Size and Zetapotential Measurements:

(41) The hydrodynamic diameters of polyplexes as prepared above were measured by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK) according to the SOPs distributed by Malvern. The measurements were performed at 25 C. in the specified buffer analysed by a cumulant method to obtain the hydrodynamic diameters and polydispersity indices of the polyplexes.

(42) The Zeta potential of the polyplexes 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.

(43) 7. Gel Shift Assay

(44) Furthermore, mRNA coding for luciferase (Luc-RNActive) according to SEQ ID NO: 369 was formulated with the polymers as indicated and aliquots were incubated with either heparin (100 g) or Dithiothreitol (DTT) for 15 Minutes at 37 C. Afterwards electrophoresis was done on agarose gel and the nucleic acids were visualized by ethidium bromide staining.

(45) 8. Confocal Laser Scanning Microscopy

(46) Confocal laser scanning microscopy was performed on an inverted LSM510 laser scanning microscope (Carl Zeiss, Germany) using a Plan-Apochromat 63/1.4 N.A. lens. All analyses were performed with living, nonfixed cells grown in eight-well chambered cover slides (Nunc, Germany). For the detection of Alexa Fluor 647 labelled mRNA only the light of a 633-nm helium neon laser, directed over a UV/488/543/633 beam splitter in combination with a LP 650 long pass filter was used. Life cell microscopy was performed at room temperature.

(47) For this purpose, L929 cells (2510.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. Cells were transfected with formulations containing 2 g Alexa Fluor 647 labelled mRNA in 8 chamber well slides directly before conduction of the microscopy experiment.

(48) 8. Cytokinstimulation in hPBMC

(49) 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 with 10 l of the RNA/carrier complex in X-VIVO 15 Medium (BioWhittaker). As RNA SEQ ID NO: 369 was used. The carriers were as shown above for generic formula (I). The immunostimulatory effect upon the hPBMC cells was measured by detecting the cytokine production (Interleukin-6; Tumor necrose factor alpha, Interferon alpha). Therefore, ELISA microtiter plates (Nunc Maxisorb) were incubated over night (o/n) with binding buffer (0.02% NaN.sub.3, 15 mM Na.sub.2CO.sub.3, 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, 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 1PBS, 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).

(50) 9. Transfection of HeLa Cells:

(51) 4 g RNA stabilized luciferase mRNA (Luc-RNActive) according to SEQ ID NO: 85 were mixed in molar ratios as indicated with the respective polymer (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 table 1 above.

(52) 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 50 l of the RNA/carrier complex solution were mixed with 250 l serum free or FCS containing medium (as indicated in the provided table) and added to the cells (final RNA concentration: 13 g/ml). Prior to addition of the serum free 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-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 M luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).

(53) 10. Expression of Luciferase In Vivo:

(54) 10 g mRNA coding for luciferase (Luc-RNActive) according to SEQ ID NO: 369 were mixed in a molar ratio of 1:250 (RNA:polymeric carrier) with the respective carrier (according to formula I), thereby forming a complex. Afterwards the resulting solution was adjusted with Ringer Lactate solution to a final volume of 100 l and incubated for 30 minutes at room temperature, yielding a solution with a 0.1 g/l concentration of complexed RNA.

(55) 100 l (20 l) of this solution was administrated intradermally (ear pinna or back) or intramuscularly (m. tibialis) to 7 week old BALB/c mice. After 24 h the mice were sacrificed and the samples (ear, skin from the back or muscle) were collected, frozen at 78 C. and lysed for 3 Minutes at full speed in a tissue lyser (Qiagen, Hilden, Germany). Afterwards 600 l 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 MgSO4, 5 mM ATP, 62.5 M luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).

(56) 11. Results:

(57) 11.1. DLS/Zetasizer Determinations:

(58) The size and -potential of the polymeric carrier cargo complexes according to the invention were evaluated in triplicates by dynamic light scattering (DLS) and laser-doppler electrophoresis and compared to different polymeric carrier cargo complexes known in the state of the art. Table 1 summarizes the cumulant diameters and the -potential of the polymeric carrier cargo complexes.

(59) TABLE-US-00007 Polymeric Cumulant Cumulant carrier diameter diameter - cargo in water in ringer potential complex N/P (nm) lactate (nm) (mV) PB19 6 58 2 81 3 9 mV PEI 10 88 4 110 2 +20 mV RPC like 5 170 8 >1000 +39 mV polymer CH6R4H6C

(60) The cumulant diameters of the polymeric carrier cargo complexes formed by polymers according to the invention and mRNA coding for luciferase (Luc-RNActive) according to SEQ ID NO: 369 showed that small, uniform complexes were formed. These complexes are stable against agglomeration in salt containing buffer and are less than 100 nm in size. In contrast polymeric carrier cargo complexes according to the RPC procedure are unstable in salt containing buffers, forming large aggregates. The polymeric carrier cargo complexes according to the invention also form complexes of low -potential, which is linked to a low tendency in binding components of the serum, and therefore have a low tendency for opsonisation.

(61) 11.2. Confocal Microscopy:

(62) The transfection of L929 cells with AlexaFlour647 aminoallyl-labelled RNA complexed with the inventive polymer PB19 after 5 minutes already led to detectable complexes in the cell. The results are shown in FIGS. 2 and 3. As can be seen in FIG. 2, confocal microscopy of L929 cells 5 minutes after transfection with fluorescence labelled RNA (SEQ ID NO: 369) complexed with the inventive polymer PB19 in a molar ratio of 1:500 showed that already 5 minutes after transfection several complexes are detectable in the cells. After 1 h confocal microscopy of L929 cells 1 h after transfection with fluorescence labelled RNA complexed with the inventive polymer PB19 in a ratio of 1:500 revealed that after transfection most of the particles were taken up in the cells (see FIG. 3)

(63) 11.3. Stability Towards Electrostatic Displacement:

(64) Since the complexes of the present invention, particularly the polymers according to formula (I) are unique with respect to their composition and their surface charge, unexpected results could be observed in gel shift assays. Normally, it is determined, as to whether a polymer condenses the nucleic acid and thus prevents the nucleic acid to migrate in an electrical field. If a complex partner is added, which exhibits a stronger affinity for the cationic polymer than the nucleic acid, the nucleic acid is displaced from the complex and again can migrate in an electrical field. For this purpose, PEI may be used, which is known to exhibit extremely strong complexes with nucleic acid.

(65) In a gel shift assay to examine the strength of complex binding (see FIG. 4) it could be shown that the addition of the anionic polymer heparin can not displace the RNA from the inventive polymeric carrier cargo complex because it still migrates in the gel. This indicates that the complex binding is so strong that a competitive complex partner cannot displace the RNA from the inventive polymeric carrier cargo complex.

(66) In a further a gel shift assay to examine the strength of complex binding (see FIG. 5) it could be shown that the addition of the anionic polymer heparin or the reducing agent DTT alone cannot display the RNA from the inventive polymeric carrier cargo complex with the polymer according to the invention (PB19). Only together they are able to display the RNA from the complex (lane 7). This indicates that the complex binding is so strong that neither a competitive complex partner nor a reducing agent can displace the RNA from the inventive polymeric carrier cargo complex.

(67) Contrary to PEI complexes, the RNA is not released from the inventive polymeric carrier cargo complex upon addition of heparin. Only a combination of heparin and DTT releases the RNA. It is to be noted that DTT reduces disulfide bonds and thus destroys the conjugate. This imitates in vivo conditions, where the reducing conditions in the cell releases the RNA from the complex.

(68) In comparison thereto, gel shift assays with PEI complexes to examine the strength of complex binding show that the addition of the anionic polymer heparin alone cannot displace the RNA from the complex with polymers used according to the present invention (PB22). In contrast mRNA could be readily displayed by heparin from PEI complexes (see FIG. 6).

(69) 11.4. Expression of Luciferase in HeLa Cells:

(70) The expression of luciferase in HeLa cells was determined using a complex of a polymer according to formula (I) herein and an mRNA according to SEQ ID NO: 369 (mRNA coding for luciferase (luc-RNActive) with the PB19 carrier (molar ratio of RNA:PB19 1:1000, 1:500, 1:100). These formulations of mRNA coding for luciferase (luc-RNActive) with the PB19 carrier (molar ratio of RNA:PB19 1:1000, 1:500, 1:100) lead to expression of luciferase independently of the presence of serum containing medium. These results are unexpected because serum containing medium leads in general to a loss of transfection efficiency.

(71) 11.5. Expression of Luciferase In Vivo:

(72) Expression of luciferase in BALB/c mice was determined after intradermal injection. As can be seen in FIG. 9a, formulations of mRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 369) with the PB19 polymer lead to expression of luciferase in the dermis of female BALB/c mice. Other transfection reagents known in the art (PEI and Lipofectamine 2000) did not show any expression of the Luciferase protein.

(73) Furthermore, expression of luciferase in BALB/c mice after intramuscular injection was determined. As a result (see FIG. 9b), formulations of mRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 369) with the PB19 polymer lead to expression of luciferase in the m. tibialis of female BALB/c mice. Other transfection reagents known in the art (PEI and Lipofectamine 2000) did not show any expression of the Luciferase protein.

(74) Additionally, expression of luciferase in BALB/c mice after intradermal injection of different formulations was determined. As a result (see FIG. 10), formulations of mRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 369) with two different polymers according to the present invention (polymers PB19 and PB48) lead to expression of luciferase in the dermis of female BALB/c mice. The polymeric carrier cargo complex formed by a peptide according to RPC CH6R4H6C (without PEGylation) and RNA in a molar ratio of 2500:1 procedure showed no expression of luciferase after intradermal injection of the complexed RNA.

(75) 11.6 Cytokine Stimulation in hPBMC

(76) Cytokine stimulation, particularly hIL-6 cytokine secretion in hPBMCs was measured. As a result (see FIG. 13) it could be shown that the complexes according to the invention consisting of RNA (SEQ ID NO: 369) and polymers according to the invention (PB19 and PB22) do not induce the secretion of hIL-6 in hPBMCs in contrast to complexes consisting of RNA (SEQ ID NO: 369) and cationic peptides (H.sub.3R.sub.9H.sub.3) or a combination of cationic peptides (H.sub.3R.sub.9H.sub.3) and PEGylated peptides (E9 or R9) (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates). E9-PEG5k: HO-PEG.sub.5000-EEEEEEEEE E9-PEG3k: HO-PEG.sub.3000-EEEEEEEEE R9-PEG3k: HO-PEG.sub.3000-RRRRRRRRR PB19: HO-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.5S-PEG.sub.5000-OH PB22: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHCS).sub.5S-PEG.sub.5000-OH

(77) Furthermore, hTNFa cytokine secretion in hPBMCs was measured. The results show (see FIG. 14), that the complexes according to the invention consisting of RNA (SEQ ID NO: 369) and polymers according to the invention (PB19 and PB22) do not induce the secretion of hTNF in hPBMCs in contrast to complexes consisting of RNA (SEQ ID NO: 369) and cationic peptides (H.sub.3R.sub.9H.sub.3) or such complexes coated with PEGylated peptides (E9 or R9) (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates). E9-PEG5k: HO-PEG.sub.5000-EEEEEEEEE E9-PEG3k: HO-PEG.sub.3000-EEEEEEEEE R9-PEG3k: HO-PEG.sub.3003-RRRRRRRRR PB19: HO-PEG.sub.5000-S(SCHHHRRRRHHHCS).sub.5S-PEG.sub.5000-OH PB22: HO-PEG.sub.5000-S(SCHHHHHHRRRRHHHHHHCS).sub.5S-PEG.sub.5000-OH

(78) Moreover, hIFNa cytokine secretion in hPBMCs was determined in a comparison of cytokine stimulating properties of complexes according to the invention consisting of RNA (SEQ ID NO: 369) and polymers according to the invention (PB19) to state of the art transfection reagents like Lipofectamin 2000 or PEI. As a result both Complexation with Lipofectamin 2000 and PEI lead to a high amount of secretion of hIFNa, whereas the complex according to the invention consisting of RNA (SEQ ID NO: 369) and polymers according to the invention (PB19 500) did not.

(79) Furthermore, hTNFa cytokine secretion in hPBMCs was measured in a comparison of cytokine stimulating properties of complexes according to the invention consisting of RNA (SEQ ID NO: 369) and polymers according to the invention (PB19) to state of the art transfection reagents like Lipofectamin 2000 or PEI. As a result (see FIG. 15) Complexation with Lipofectamine 2000 leads to a high amount of secretion of hTNF, whereas complexation with PEI or polymers according to the invention (PB19 500) did not.