RNA formulation for immunotherapy

Abstract

The present invention is in the field of immunotherapy, in particular tumor immunotherapy. The present invention provides pharmaceutical formulations for delivering RNA to antigen presenting cells such as dendrite cells (DCs) in the spleen after systemic administration. In particular, the formulations described herein enable to induce an immune response after systemic administration of antigen-coding RNA.

Claims

1. A pharmaceutical composition comprising nanoparticles that comprise: (i) at least one cationic lipid, and (ii) at least one RNA molecule encoding at least one antigen, wherein, at physiological pH, an overall charge ratio of positive charges to negative charges of the nanoparticles is between 1:1.2 (0.83) and 1:2 (0.5), wherein the positive charges are contributed by the at least one cationic lipid, and the negative charges are contributed by the at least one RNA molecule; a polydispersity index of the nanoparticles is 0.5 or less as measured by dynamic light scattering; and the nanoparticles have an average diameter in the range of from about 250 nm to about 700 nm as measured by dynamic light scattering.

2. The pharmaceutical composition of claim 1, wherein at physiological pH the overall charge ratio of positive charges to negative charges of the nanoparticles is between 1.6:2 (0.8) and 1:2 (0.5).

3. The pharmaceutical composition of claim 1, wherein at physiological pH the overall charge ratio of positive charges to negative charges of the nanoparticles is between 1.6:2 (0.8) and 1.1:2 (0.55).

4. The pharmaceutical composition of claim 1, wherein at physiological pH the overall charge ratio of positive charges to negative charges of the nanoparticles is 1.3:2 (0.65).

5. The pharmaceutical composition of claim 1, wherein the polydispersity index of the nanoparticles is 0.4 or less as measured by dynamic light scattering.

6. The pharmaceutical composition of claim 1, wherein the polydispersity index of the nanoparticles is 0.3 or less as measured by dynamic light scattering.

7. The pharmaceutical composition of claim 1, wherein the nanoparticles have a zeta potential of from 0 mV to −50 mV.

8. The pharmaceutical composition of claim 1, wherein the nanoparticles have a zeta potential of from −10 mV to −30 mV.

9. The pharmaceutical composition of claim 1, wherein the nanoparticles have an average diameter in the range of from about 250 nm to about 550 nm as measured by dynamic light scattering.

10. The pharmaceutical composition of claim 1, wherein the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

11. The pharmaceutical composition of claim 1, wherein the at least one cationic lipid comprises DOTMA.

12. The pharmaceutical composition of claim 1, wherein the at least one cationic lipid forms a complex with and/or encapsulates the at least one RNA molecule.

13. The pharmaceutical composition of claim 1, wherein the at least one cationic lipid is comprised in a vesicle encapsulating the at least one RNA molecule.

14. The pharmaceutical composition of claim 1, wherein the nanoparticles further comprise at least one helper lipid.

15. The pharmaceutical composition of claim 14, wherein the at least one helper lipid is a neutral lipid.

16. The pharmaceutical composition of claim 14, wherein the at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2-diolcoyl-sn-glycero-3-phosphocholine (DOPC).

17. The pharmaceutical composition of claim 14, wherein the at least one helper lipid comprises DOPE.

18. The pharmaceutical composition of claim 14, wherein a molar ratio of the at least one cationic lipid to the at least one helper lipid is from 9:1 to 3:7.

19. The pharmaceutical composition of claim 18, wherein the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1.

20. The pharmaceutical composition of claim 18, wherein the molar ratio of the at least one cationic lipid to the at least one helper lipid is about 2:1.

21. The pharmaceutical composition of claim 14, wherein the at least one cationic lipid is DOTMA and the at least one helper lipid is DOPE.

22. The pharmaceutical composition of claim 21, wherein the molar ratio of DOTMA to DOPE is about 2:1.

23. The pharmaceutical composition of claim 14, wherein: at physiological pH the overall charge ratio of positive charges to negative charges of the nanoparticles is between 1:1.2 (0.83) and 1:2 (0.5); the nanoparticles have an average diameter in the range of from about 250 nm to about 550 nm as measured by dynamic light scattering; and the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 9:1 to 3:7.

24. The pharmaceutical composition of claim 23, wherein: at physiological pH the overall charge ratio of positive charges to negative charges of the nanoparticles is 1.3:2 (0.65); the nanoparticles have an average diameter in the range of from about 300 nm to about 500 nm as measured by dynamic light scattering; and the at least one cationic lipid is DOTMA and the at least one helper lipid is DOPE, and the molar ratio of DOTMA to DOPE is about 2:1.

25. The pharmaceutical composition of claim 1, wherein the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 8:2 to 3:7, and wherein the charge ratio of positive charges in DOTMA to negative charges in the at least one RNA molecule is 1.6:2 (0.8) to 1:2 (0.5).

26. The pharmaceutical composition of claim 25, wherein the lipoplexes comprise DOTMA and DOPE in a molar ratio of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the at least one RNA molecule is about 1.3:2 (0.65).

27. The pharmaceutical composition of claim 1, wherein the nanoparticles are lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 8:2 to 3:7, and wherein the charge ratio of positive charges in DOTMA to negative charges in the at least one RNA molecule is 1.6:2 (0.8) to 1:2 (0.5).

28. The pharmaceutical composition of claim 27, wherein the lipoplexes comprise DOTMA and Cholesterol in a molar ratio of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the at least one RNA molecule is about 1.3:2 (0.65).

29. The pharmaceutical composition of claim 1, wherein the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 8:2 to 3:7, and wherein the charge ratio of positive charges in DOTAP to negative charges in the at least one RNA molecule is 1.6:2 (0.8) to 1:2 (0.5).

30. The pharmaceutical composition of claim 29, wherein the lipoplexes comprise DOTAP and DOPE in a molar ratio of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTAP to negative charges in the at least one RNA molecule is about 1.3:2 (0.65).

31. The pharmaceutical composition of claim 1, wherein the nanoparticles are produced by a process comprising a step of incubating the at least one RNA molecule with bivalent cations prior to incorporation into said nanoparticles and/or by incubating the at least one RNA molecule with monovalent ions prior to incorporation into said nanoparticles and/or by incubating the at least one RNA molecule with one or more buffers prior to incorporation into said nanoparticles.

32. The pharmaceutical composition of claim 1, wherein the at least one antigen is a disease-associated antigen or elicits an immune response against a disease-associated antigen or cells expressing a disease-associated antigen.

33. The pharmaceutical composition of claim 1, wherein the at least one antigen is a tumor antigen or an antigen from an infectious agent.

34. The pharmaceutical composition of claim 1, wherein the at least one RNA molecule is or comprises mRNA.

35. The pharmaceutical composition of claim 1, wherein the at least one RNA molecule encoding at least one antigen comprises an unmasked poly-A sequence.

36. The pharmaceutical composition of claim 35, wherein the unmasked poly-A sequence has a length of about 120 nucleotides.

37. The pharmaceutical composition of claim 1, wherein the at least one RNA molecule encoding at least one antigen comprises a 3′UTR.

38. The pharmaceutical composition of claim 1, wherein the at least one RNA molecule encoding at least one antigen further comprises a 5′-cap.

39. The pharmaceutical composition of claim 38, wherein the 5′ cap comprises: ##STR00002## wherein: R.sub.1 and R.sub.2 are each independently hydroxyl or methoxy; and W, X, and Y are each independently selected form the group consisting of oxygen, sulfur, selenium and BH.sub.3.

40. The pharmaceutical composition of claim 39, wherein: (a) R.sub.1 and R.sub.2 are OH, and W, X, and Y are oxygen; (b) one of R.sub.1 and R.sub.2 is OH, the other R.sub.1 and R.sub.2 is methoxy, and W, X, and Y are oxygen; (c) R.sub.1 and R.sub.2 are OH, X is sulfur, and W and Y are oxygen; or (d) R.sub.2 is OH, R.sub.1 is methoxy, one of W, X, and Y is sulfur, and the other two of W, X, and Y are oxygen.

41. The pharmaceutical composition of claim 39, wherein R.sub.2 is OH, R.sub.1 is methoxy, X is sulfur, and W and Y are oxygen.

42. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, excipients, and/or adjuvants.

43. A method of treating or preventing a disease involving an antigen in a subject comprising administering to the subject the pharmaceutical composition of claim 1, wherein the antigen is a disease-associated antigen or elicits an immune response against a disease-associated antigen or cells expressing a disease-associated antigen, whereby the disease in the subject is treated or the subject is prevented from developing the disease.

44. A pharmaceutical composition comprising nanoparticles that comprise: (i) at least one cationic lipid, (ii) at least one RNA molecule encoding at least one antigen, and (iii) at least one helper lipid, wherein, at physiological pH, an overall charge ratio of positive charges to negative charges of the nanoparticles is 1.3:2 (0.65), wherein the positive charges are contributed by the at least one cationic lipid, and the negative charges are contributed by the at least one RNA molecule; a polydispersity index of the nanoparticles is 0.5 or less as measured by dynamic light scattering; the nanoparticles have an average diameter in the range of from about 250 nm to about 700 nm as measured by dynamic light scattering; the at least one cationic lipid is DOTMA; the at least one helper lipid is DOPE; and a molar ratio of DOTMA to DOPE is about 2:1.

Description

FIGURES

(1) FIG. 1: Size of F4/RNA lipoplexes at different DOTMA/RNA charge ratios (2/1, 1/1, 1/2, 1/4) in water (a), PBS (b) and in PBS after addition of 2.2 mM CaCl.sub.2 (c) and 22 mM CaCl.sub.2 (d).

(2) FIG. 2: Particle sizes of DOTMA/Chol liposomes (F5) and lipoplexes at different buffers and DOTMA/RNA charge ratios 1/1 and 2/1 (positive excess).

(3) FIG. 3: Mean size of F5/RNA lipoplexes at charge ratios (1/1) and (1/2) after compaction of RNA using different amounts of CaCl.sub.2.

(4) FIG. 4: Overview of selected results from physico-chemical characterization of RNA lipoplexes with DOTMA/DOPE liposomes. The x-axis gives the charge ratio between DOTMA and RNA. Top: particle size from PCS measurements, middle: polydispersity index, bottom: zeta potentials of the same formulations. The lines have been introduced to guide the eye.

(5) FIG. 5: (a) Mean size of F4/Luc-RNA lipoplexes at the charge ratio (1/2) in water and after addition of concentrated buffer to PBS (1×), sodium chloride (150 mM), glucose (5%) or phosphate buffered glucose. In contrast to the 1/1-ratio, which leads to aggregation under all buffer conditions (not shown here), the particle sizes of the lipoplexes at the 1/2 ratio were approximately 220 nm. (b) Polydispersity of size ranged from 0.23 to 0.34 indicating colloidal stability.

(6) FIG. 6: (a) Mean size of F4/RNA lipoplexes at selected DOTMA/RNA charge ratios. Particle sizes of lipoplexes with charge ratios between 1:1.8 and 1:1.4 were approximately 160 nm. With decreasing negative excess (charge ratio 1:1.2) particle size was determined to 183 nm. (b) All tested charge ratios leads to lipoplexes with small polydispersity indices less than 0.2.

(7) FIG. 7: (a) Mean Size of DOTMA/DOPE liposomes (1:2) in water without extrusion (F4-raw), after extrusion using a polycarbonate membrane with a pore diam. of 400 nm (F4-400), 200 nm (F4-200), 100 nm (F4-100) or 50 nm (F4-50). Corresponding lipoplexes with a DOTMA/RNA charge ratio of 1/2 in water (2:) and in PBS buffer (3:). (b) Polydispersity of size of the lipoplexes with extruded liposomes ranged from 0.10 to 0.28. However, lipoplexes formed by un-extruded liposomes also showed a sufficiently narrow size distribution.

(8) FIG. 8: Mean size (a) and Polydispersity Index (b) of DOTMA/DOPE liposomes (F4) determined before lyophilization and after lyophilization and reconstitution using water.

(9) FIG. 9: Particle size of liposomes with different DOTMA/DOPE ratios. For liposomes with high DOPE (90%) fraction, the particles are unstable in PBS and aggregate.

(10) FIG. 10: Particle size of lipoplexes with liposomes comprising different DOTMA/DOPE ratios. With the DOTMA/DOPE ratio from 9/1 to 4/6, the lipoplexes have defined particle sizes (<300 nm) with low PI values (<0.2). With higher DOPE fraction, larger particle sizes with high PI values are obtained.

(11) FIG. 11: Luciferase activities in vivo and ex vivo after injection into BALB/c mice of luciferase-RNA (20 μg) complexed with different amounts of F4 liposomes to yield F4:RNA ratios of 4.8:1, 2.4:1, 1.2:1, 1.2:2, 1.2:4.

(12) FIG. 12: Distribution of total luciferase signal among organs derived from the experiment depicted in FIG. 11.

(13) FIG. 13: Luciferase activities in vivo and ex vivo after injection into BALB/c mice of Luciferase-RNA (20 μg) complexed with F11 or F12 liposomes.

(14) FIG. 14: Luciferase activities in vivo and ex vivo after injection into BALB/c mice of Luciferase-RNA (20 μg) complexed with F2 or F5 liposomes.

(15) FIG. 15: Quantification of luciferase activities in spleens of mice after injection of Luciferase-RNA (20 μg) diluted in 1×PBS (A) or undiluted in water (B and C) complexed with F4 liposomes diluted in 1×PBS (B) or undiluted in water (A and C) with an F4:RNA ratio of 1.2:2. The final PBS concentrations of all complexes were set to 1×PBS.

(16) FIG. 16: Quantification of luciferase activities in spleens of mice after injection of Luciferase-RNA (20n) precomplexed with 0.125 or 1 mM CaCl.sub.2 or without precomplexation and mixed with F4 liposomes with an F4:RNA ratio of 1.2:2.

(17) FIG. 17: Quantification of luciferase activities in spleens of mice after injection of Luciferase-RNA (20 μg) or F4 liposomes diluted in 1×PBS or 154 mM NaCl and mixed with an F4:RNA ratio of 1.2:2.

(18) FIG. 18: Quantification of luciferase activities in spleens of mice after injection of Luciferase-RNA (20 μg) precomplexed with 1-4 mM CaCl.sub.2 and mixed with F4 liposomes with an F4:RNA ratio of 1.2:2 using 154 mM NaCl instead of 1×PBS as dilution buffer.

(19) FIG. 19: (A) Luciferase-RNA (5 μg) was incubated in 25 or 50% mouse serum for 30 min. and then electroporated into human monocyte derived immature DCs. Luciferase activity was assessed 18 h later via standard in vitro luciferase assay. (B) Luciferase-RNA (20 μg) was complexed via standard protocol with F4 liposomes with an F4:RNA ratio of 1.2:2 and then incubated in the presence or absence of 50% mouse serum for 30 min. BALB/c mice were injected intravenously with these formulations and luciferase activities in vivo were quantified from spleens of mice.

(20) FIG. 20: Assessment of the uptake of Cy5-RNA or F4-rho by cell populations in spleen after injection into BALB/c mice of Cy5-RNA (40 μg) complexed with F4 liposomes labeled with Rhodamine (F4-rho) (1.2:2; Liposome:RNA).

(21) FIG. 21: Assessment of the (A) maturation status of dendritic cells (revealed by upregulation of CD86 and CD40) and (B) serum concentrations of IFNa and TNFa after injection into C57BL/6 mice of HA-RNA (40 μg) complexed with F4 (1.2:2; Liposome:RNA), F4 alone or PBS (as control).

(22) FIG. 22: Assessment of the (A) frequencies of antigen specific CD8.sup.+ T cells and (B) memory recall responses after immunization of C57BL/6 mice with SIINFEKL-RNA (20 or 40 μg) complexed with F4 liposomes at different liposome:RNA ratios.

(23) FIG. 23: Kaplan-Meier survival curves of C57BL/6 mice which received three intravenous immunizations of SIINFEKL-RNA (40 μg) complexed with F4 liposomes with an F4:RNA ratio of 1.2:2 or were left untreated and into which were injected 2×10.sup.5 B16-OVA tumor cells s.c. into the flanks.

(24) FIG. 24: Individual tumor growth after s.c. inoculation of 2×10.sup.5B16-OVA tumor cells into the flanks of C57/Bl6 mice which received seven intravenous immunizations of SIINFEKL-RNA (40 μg) complexed with F4 or F12 liposomes with an F4:RNA ratio of 1.2:2. Liposomes alone without SIINFEKL-RNA were used as control treatment.

(25) FIG. 25: Kaplan-Meier survival curves after s.c. inoculation of 2×10.sup.5 B16-OVA tumor cells into the flanks of C57/Bl6 mice which received seven intravenous immunizations of SIINFEKL-RNA (40 μg) complexed with F4 or F12 liposomes with an F4:RNA ratio of 1.2:2. Liposomes alone without SIINFEKL-RNA were used as control treatment.

(26) FIG. 26: Luciferase activities in vivo and ex vivo after injection into BALB/c mice of luciferase-RNA (20 μg) complexed with different amounts of F5 liposomes to yield F5:RNA ratios of 4.8:1, 2.4:1, 1.2:1, 1.2:2, 1.2:4.

(27) FIG. 27: Distribution of total luciferase signal among organs derived from the experiment depicted in FIG. 26.

(28) FIG. 28: Preformulation of RNA and reconstitution of RNA-lipoplex solution.

(29) FIG. 29: Results of DLS measurements of RNA lipoplexes reconstituted according the clinical formulation protocol. Limited spread of received lipoplex particle sizes demonstrates the robustness of the procedure of mixing.

(30) FIG. 30: Particle size and Polydispersity Index of 1:2 lipoplexes of extruded and non extruded liposomal precursors.

(31) FIG. 31: Luciferase activities in vivo after injection into BALB/c mice of luciferace-RNA (20 μg) complexed with small or big liposomes in PBS to achieve lipoplexes different in size.

(32) FIG. 32: Quantification of luciferase activities in spleens of mice after injection of Luciferase-RNA lipoplexes different in size. Lager lipoplexes, assembled from larger liposomes, have higher activity, independent from the lipid composition of the liposomes.

(33) FIG. 33: Lipoplexes formed by using NaCl and PBS buffer in ‘normal’ and 10× concentrated form. In the latter case, a 10-fold lower volume was added to obtain the same final concentration. All lipoplexes have about the same size but those from concentrated solutions are a bit smaller.

(34) FIG. 34: Activity (luc expression) of the lipoplexes measured in FIG. 33. As a trend, the lipoplexes from non-concentrated buffers are higher in activity. Treatment with normal saline yields highest activity.

(35) FIG. 35: Lipoplexes formed after addition of the NaCl to the RNA at different concentrations. The final NaCl concentration was in all cases the same, as from the concentrated solutions lower volumes were added. As a trend, the lipolex size increases with decreasing concentration of the added NaCl solution. As larger lipoplexes are higher in activity than smaller ones, use of 0.9% NaCl (150 mM) is considered to result in the best activity.

(36) FIG. 36: Size (Zave) and Polydispersity Index (PI), for lipoplexes with different mixing ratios (DOTMA/nucleotide ratios), directly after reconstitution, and after 2 h and 24 h.

(37) FIG. 37: Results of DLS measurements of RNA lipoplexes with different charge ratios tested in vivo.

(38) FIG. 38: Quantification of luciferase activities in spleens of mice after injection of Luciferase-RNA lipoplexes different in size.

EXAMPLES

(39) The techniques and methods used herein are described herein or carried out in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods including the use of kits and reagents are carried out according to the manufacturers' information unless specifically indicated.

Example 1: Materials and Methods

(40) Liposome Preparation

(41) Manufacturing of liposomes was performed by different protocols. The ‘film method’ or ‘ethanol injection’ was used for liposome preparation. For the film method, the lipids were dissolved in chloroform and put in appropriate amounts into a round bottom flask. The organic solvent was evaporated in a rotary evaporator and the dry film was reconstituted with water or buffer/excipient solution by gently shaking of the flask. Typically, a total lipid concentration of 5 mM was selected. For ethanol injection, the lipids were dissolved at suitable molar ratios in ethanol to a total concentration in the range of 100-400 mM. The ethanol solution was injected under stirring into water or the aqueous solution of buffers/excipients. The size of the liposomes was adjusted by extrusion across polycarbonate membranes of different pore size (50-400 nm), and/or they were filtered through commercially available sterile filters of 220-450 nm pore size, or filters for clinical use with other pore sizes (1 μm-5 μm) were used (Sartorius, Göttingen, Germany, Millipore, Schwalbach, Germany).

(42) The final lipid concentration in the aqueous phase was between 5 mM and 25 mM. Lipid composition was controlled by HPLC analysis. Particle size and zeta potential were determined by dynamic light scattering.

(43) Lipoplex Formation

(44) Lipoplex formation was performed by different protocols. The detailed procedure is given with the individual experiments. For several experiments, direct incubation of RNA solutions with liposome solutions in water or in the presence of buffers or excipients was performed. Lipoplexes could also be formed by mixing of lipid solutions in ethanol with RNA solutions in water or aqueous buffer/excipient solutions. The selected preparation protocol depended on the desired particle characteristics and biological application and is further described with the respective experiments.

(45) PCS Measurements

(46) Particle size and zeta potential measurements were performed on a 380 ZLS submicron particle/zeta potential analyzer (PSS Nicomp, Santa Barbara, Calif.). Size was determined by Photon correlation spectroscopy (PCS) at a scattering angle of 90° with an equilibration time of 2 min and run times of 15 min. Auto correlation was performed using the intensity-weighted Gaussian analysis, which gives information about the mean diameter of the bulk population and the polydispersity index (PI).

(47) Zeta Potential

(48) Zeta potential was measured in water using electric field strength of 5 V/cm and an electrode spacing of 0.4 cm. The electrostatic mobility was converted to the zeta potential using the Helmholtz-Smoluchowski equation. All measurements were carried out at a temperature of 23° C.

(49) Field-Flow-Fractionation

(50) Asymmetrical Flow FFF (AF4) was performed using the Eclipse 3+ system equipped with a long channel (275 mm length) and the triple-angle MALS light scattering detector miniDAWN TREOS (Wyatt Technologie, Dernbach, Germany) using the following hardware/parameters:

(51) Membrane: 10 kD regenerated cellulose (Microdyn Nadir, Wiesbaden, Germany)

(52) Spacer: 250 μm spacer (wide 21.5 mm)

(53) Solvent: 10 mM NaNO3

(54) Detector flow: 1.0 mL/min

(55) Focus flow: 1.5 mL/min

(56) Injektion flow: 0.2 mL/min

(57) Cross flow gradient: 4 mL/min (fixed for 15 min, than 4 mL/min to 0.1 mL/min in 20 min).

(58) Animals

(59) C57BL/6 and BALB/c mice were from Jackson Laboratories. Age (8-10 weeks old) and sex (female) matched animals were used throughout the experiments.

(60) Cells and Cell Lines

(61) B16-OVA is a B16-F10 melanoma cell line expressing the chicken ovalbumin gene (OVA). Human monocyte derived immature DCs (iDC) were differentiated from purified CD14.sup.+ monocytes in the presence of IL-4 (1000 U/ml) and GM-CSF (1000 U/ml) for 5 days.

(62) RNA Constructs and In Vitro Transcription

(63) All plasmids for in vitro transcription of naked antigen-encoding RNA were based on the pST1-2hBgUTR-A120 backbone which feature a 3′ human β-globin UTR (hBgUTR) and a poly(A) tail of 120 nucleotides and allow generation of pharmacologically improved in vitro transcribed RNA. The SIINFEKL construct contains aa 257-264 of chicken OVA. HA construct was a codon optimized partial sequence of influenza HA (aa 60-285 fused to aa 517-527; influenza strain A/PR/8/34) designed to combine major immunodominant MHC epitopes. pSTI-Luciferase-A120 (Luc) contains the firefly luciferase gene (15). RNA was generated by in vitro transcription. Labeling of RNA with Cy5-UTP (Cy5-RNA) was conducted according to the manufacturer's instructions (Amersham Biosciences, Buckinghamshire, UK) using the HA construct as template.

(64) Preparation and Injection of Lipoplexes

(65) Unless otherwise stated, as standard protocol, RNAs and Liposomes were prediluted in 1× RNase free phosphate buffered saline (PBS) (Ambion) to a final volume of 100 μl prior to mixing. 10 minutes after mixing of diluted RNA and liposome, 200 μl lipoplex solution was injected per mouse intravenously. For some experiments, PBS was replaced with 154 mM RNease free NaCl (Ambion)

(66) Flow Cytometric Analysis

(67) Monoclonal antibodies for flow cytometry were from BD Pharmingen. Hypotonicly lysed blood samples were incubated at 4° C. with specific mABs. Spleen cells were obtained by digestion with collagenase (1 mg/ml; Roche). Quantification of SIINFEKL-specific CD8.sup.+ cells with H-2 K.sup.b/SIINFEKL tetramer (Beckman-Coulter) was previously described. Flow cytometric data were acquired on a FACS-Canto II analytical flow cytometer and analyzed by using FlowJo (Tree Star) software.

(68) Electroporation

(69) 50 μl of RNA solution was electroporated into iDCs with electoporation parameters of 270V and 150 μF using BioRad electroporator.

(70) In Vivo Bioluminescence Imaging (BLI)

(71) Uptake and translation of Luc-RNA were evaluated by in vivo bioluminescence imaging using the IVIS Lumina imaging system (Caliper Life Sciences). Briefly, an aqueous solution of D-luciferin (150 mg/kg body weight) (BD Biosciences) was injected i.p. 6 h after administration of RNA lipoplexes. 5 min thereafter, emitted photons were quantified (integration time of 1 min). In vivo bioluminescence in regions of interest (ROI) were quantified as average radiance (photons/sec/cm.sup.2/sr) using IVIS Living Image 4.0 Software. The intensity of transmitted light originating from luciferase expressing cells within the animal was represented as a grayscale image, where black is the least intense and white the most intense bioluminescence signal. Grayscale reference images of mice were obtained under LED low light illumination. The images were superimposed using the Living Image 4.0 software.

(72) ELISA

(73) Mouse IFN-α (PBL) and TNFa (eBioscience) was detected in mouse sera using standard ELISA assay according to manufacturer's instructions.

(74) Tumor Experiments

(75) To determine protective immunity, mice received three immunizations. Thereafter, 2×105 B16-OVA tumor cells were inoculated s.c. into the flanks of C57BL/6 mice. For assessment of therapeutic immunity, first same numbers of tumor cells were inoculated. Immunizations were then initiated after tumors had reached a diameter of 2 to 3 mm. Tumor sizes were measured every three days. Animals were sacrificed when the diameter of the tumor exceeded 15 mm.

Example 2: Effect of Buffers/Ions on Particle Sizes and PI of RNA Lipoplexes

(76) Lipoplexes of liposomes and RNA at different charge ratios +/− between the cationic (positively charged) lipid DOTMA and the negatively charged RNA were prepared. The physiochemical characteristics of the liposomes were investigated by dynamic light scattering (PCS) and zeta potential measurements.

(77) The use of buffer which is often necessary for pharmaceutical applications and ions can lead to aggregation of lipoplexes which makes them unsuitable for parenteral application to patients. In order to evaluate these effects on the average diameter of lipoplexes, the particle characteristics of lipoplexes of DOTMA/DOPE (F4) liposomes [DOTMA/DOPE (1:1 mol:mol)] and RNA at different charge ratios were determined under four buffer conditions, namely, water, PBS buffer, PBS plus 2.2 mM CaCl.sub.2, and PBS plus 22 mM CaCl.sub.2. For the measurements, briefly, lipoplexes were formed by adding of RNA to preformed liposomes, subsequently the buffers were added. The final RNA concentration was selected to about 100 μg/ml. All other concentrations were adjusted accordingly or selected as given in the figures. Particle sizes are shown in FIG. 1. The DOTMA/RNA charge ratio is given on the x-axis of each chart.

(78) (a) In water, lipoplexes of defined particle sizes (mean size less than 300 nm), with low polydispersity indices (<0.3) were obtained. The measured particle sizes were only slightly affected by the charge ratio. However, negatively charged particles are smaller (mean size 100 to 200 nm) and more stable (PI<0.15) than uncharged particles (mean size 200 to 250 nm, PI<0.2).

(79) (b) In PBS buffer, the same effect is more prominent. Lipoplexes with a positive or neutral charge ratio form larger particles (partially stabilized by the positive charges). Lipoplexes with a neutral charge ratio are building unstable aggregates. In contrast, negatively charged lipoplexes are both stable (as indicated by a low PI<0.2) and compact with average particle sizes of 250 nm and less.

(80) (c) After addition of CaCl.sub.2 an increase in the particle sizes is observable. However, at physiological Ca.sup.++ concentrations (shown: 2.2 mM; in some cell types the physiological concentration can be up to 5 mM, rarely up to 10 mM) negatively charged particles still have defined sizes below 500 nm with a polydispersity index not exceeding 0.6. For the sample with excess positive charge the size increased almost to 1000 nm.

(81) (d) Addition of 22 mM CaCl.sub.2 to the samples b) (PBS) induced aggregation/flocculation under all conditions, supposedly due to formation of calcium phosphate particles.

(82) These results demonstrate that in buffered solutions such as i.e. in PBS buffer and/or in the presence of CaCl.sub.2, positive or neutral charge ratios are poorly suited for the production of stable liposomal formulations. The stability of lipoplexes highly depends on the charge ratio +/− between the cationic DOTMA lipid and the charged RNA. In addition, both the ionic strength of the formulation buffer and the presence of bivalent cations have strong influences on particle sizes. Under physiological conditions (i.e. pH 7.4; 2.2 mM Ca.sup.++), a negative charge ratio appears to be imperative due to the instability of neutral or positively charged lipoplexes. For lipoplexes with excess negative charge the lowest trend for aggregation was observed.

Example 3: Effect of Positive Charge on Stability of RNA Lipoplexes

(83) For an additional evaluation of a potential beneficial/detrimental effect of positive charges on the stability of lipoplexes (see e.g. FIGS. 1 b and c), particle sizes of lipoplexes of DOTMA/Chol liposomes (F5) [DOTMA/Chol (1:1 mol:mol)] and RNA with DOTMA/RNA charge ratios of 1/1 and 2/1 were measured in different buffers (see FIG. 2). For comparison, also the size of the pure liposomes was measured.

(84) In 150 mM sodium chloride as well as in PBS buffer a positive 2/1 DOTMA/RNA charge ratio leads to largely increased/aggregated particle sizes with a polydispersity index greater than 0.4. This result indicates that positive charges are not suitable to stabilize lipoplexes and that aggregation has to be expected for the positively charged lipoplexes also under physiological conditions.

Example 4: Influence of Pre-Compaction of RNA Mediated by Bivalent Cations on the Particle Size of RNA Lipoplexes

(85) To test the influence of pre-compaction of RNA using divalent cations prior to the complexation, the particle size of F5/RNA lipoplexes at charge ratios (1/1) and (1/2) were determined after compaction of the RNA with different amounts of CaCl.sub.2. Contrary to Examples 2 and 3 here the ions were added to the RNA prior to lipoplex formation. The final liposome concentration was in all cases 100 μM, and the RNA concentration was adjusted accordingly. Because for the F5/RNA 1/2 the RNA concentration was doubled, here also the CaCl.sub.2 concentration was doubled.

(86) After pre-treatment of the uncharged RNA/F5 (1:1) lipoplexes with physiological concentrations of CaCl.sub.2 (i.e. 2.2 mM), the average size of the resulting lipoplex particle is inflated (i.e. to 1.2 μm); see FIG. 3. Due to this large size, such particles are not ideally suited for pharmaceutical compositions and/or the delivery of RNA into cells. In contrast, both pre-compaction experiments with negatively charged lipoplexes and low/high concentrations (low: 0.3 mM; high: 4.4 mM) of CaCl.sub.2 produced small-sized particles of approximately 200 (350) nm.

(87) These results indicate that RNA can be precondensed with bivalent ions. Due to this precondensation step, lipoplexes with defined and compact particle sizes can be formed at negative charge ratios; aggregation or substantial increase of particle size can be prevented.

Example 5: Physico-Chemical Characterization of RNA Lipoplexes

(88) In FIG. 4, results from physico-chemical characterization of RNA lipoplexes with F4 (DOTMA/DOPE) at different charge ratios +/− between DOTMA and RNA are given. As can be seen for negatively charged lipoplexes, at +/− ratios of 1/1 and above, the particle size is constant at about 200 nm. The zeta potential decreases monotonously from +/−2/1 to 1/1, and it remains constant at higher excess negative charge. These results suggest that important particle characteristics, namely particle size and zeta potential, are invariant with excess RNA, starting from the 1/1 ratio. In this range, colloidal stable particles of well-defined size can be manufactured. Similar results can also be obtained in the presence of ions and buffers (PBS).

Example 6: Effect of Buffer Composition on Stability/Particle Size of Negatively Charged RNA Lipoplexes

(89) The stability of lipoplexes in different buffers was further investigated to detail. To test if an excess of negative charge leads to colloidal stable lipoplexes in potential relevant buffer systems, particle sizes of F4/Luc-RNA lipoplexes at the charge ratio (1/2) in water and after addition of concentrated buffer to PBS (lx), sodium chloride (150 mM), glucose (5%) or phosphate buffered glucose were determined (see FIG. 5).

(90) Under all tested conditions, particle sizes are not exceeding 300 nm with PI values of clearly less than 0.4. These results suggest that, if manufactured according to the present invention, RNA lipoplexes with a charge ratio of 1/2 (excess of negatively charged RNA) are colloidally stable under different buffer conditions.

Example 7: Correlation of Negative Charge Ratio and Particle Size/Stability

(91) The colloidal stability of the lipoplexes at the ratio between (1/1) and (1/2) was further investigated. Particle sizes of F4/RNA lipoplexes with charge ratios between 1:1.8 and 1:1.2 were measured in water; see FIG. 6.

(92) These results suggest that in the range of the tested charge ratios the particle size of lipoplexes are invariant to minor changes in excess RNA. In connection with the tested (negative) charge ratios of 1:1.2 to 1:1.8, particles sizes are generally in the 100 to 200 nm range with PI values of less than 0.2.

Example 8: Effect of Extrusion on Mean Particle Size and PI Values of RNA Lipoplexes

(93) In this experiment it is shown that lipoplexes of different size can be produced. In order to determine the effect of an additional extrusion step on mean particle size and PI values of liposomes or RNA lipoplexes, extrusion experiments (using a polycarbonate membrane with different pore diameters) were performed. Results from particle sizing of RNA lipoplexes with un-extruded F4 (DOTMA/DOPE) and with extruded F4 in water or PBS are shown in FIG. 7.

(94) The experiments demonstrate that, in addition to the already described size range of 200-300 nm, also larger and smaller particles can be produced. Here, as an example particles with size in the range of 400-500 nm and <100 nm were are given.

(95) Whereas non-extruded RNA lipoplexes show average particle sizes between 400 and 500 nm, extruding of RNA lipoplexes generally leads to significantly smaller particles with sizes of less than 200 nm. In contrast, the effect of extrusion on the polydispersity is marginal; both extruded and non-extruded liposomes lead to discrete, well defined particles (with PI values between 0.1 and 0.3), if complexed with RNA.

Example 9: Effect of Lyophilization on the Particle Characteristics

(96) Lipoplexes are not stable in liquid suspension for long-term storage and aggregate. Lyophilization is one technique to address this challenge. The effect of lyophilization on the particle characteristics was investigated. Particle sizes of DOTMA/DOPE liposomes (F4) were determined before lyophilization and after lyophilization and reconstitution with water (see FIG. 8).

(97) These results suggest, that the lipoplexes can be lyophilized without affection the particle characteristics.

Example 10: Effect of DOTMA/DOPE Ratio on the Particle Characteristics

(98) Liposomes and lipoplexes with different DOTMA/DOPE ratios were manufactured. Liposomes with very high DOPE fraction (90 mol %) were unstable in PBS (FIG. 9). For lipoplexes, already at a DOPE fraction of 70 mol %, the particle size significantly increased (FIG. 10). All other compositions were stable.

Example 11: In Vivo Administration of RNA Lipoplexes

(99) BALB/c mice (n=3) were injected intravenously with Luciferase-RNA (20 μg) complexed with different amounts of F4 liposomes to yield F4:RNA ratios of 4.8:1, 2.4:1, 1.2:1, 1.2:2, 1.2:4. Luciferase activities in vivo and ex vivo were assessed via in vivo imaging 6 hours after lipoplex injection and representative mice and organ sets are shown in FIG. 11. FIG. 12 shows the distribution of total luciferase signal among organs derived from the experiment depicted in FIG. 11.

(100) F4 (DOTMA:DOPE) goes more to lungs (a little spleen) at the ratio of F4:RNA of 4.8:1, to both lungs and spleen at the ratio of F4:RNA of 2.4:1 and exclusively to spleen at ratios of F4:RNA of 1.2:1, 1.2:2, 1.2:4. Thus, neutral and anionic lipoplexes target specifically to spleen whereas cationic lipoplexes primarily target lung (wrt to protein expression). No expression in liver was detected.

(101) BALB/c mice (n=5) were injected intravenously with Luciferase-RNA (20 μg) complexed with F11 or F12 liposomes with an Fx:RNA ratio of 1.2:2 [F11: DOTMA/DOPE (1:2 mol:mol); F12: DOTMA/DOPE (2:1 mol:mol)]. Luciferase activities in vivo and ex vivo were assessed via in vivo imaging 6 hours after lipoplex injection and representative mice and organ sets are shown in FIG. 13. F4 derivatives F11 and F12 also target to spleen at an liposome:RNA ratio of 1.2:2.

(102) BALB/c mice (n=5) were injected intravenously with Luciferase-RNA (20 μg) complexed with F2 or F5 liposomes with an Fx:RNA ratio of 1:1 [F2: DOTAP/DOPE (1:1 mol:mol); F5: DOTMA/Chol (1:1 mol:mol)]. Luciferase activities in vivo and ex vivo were assessed via in vivo imaging 6 hours after lipoplex injection and representative mice and organ sets are shown in FIG. 14. At liposome:RNA ratio of 1:1, while F2 targets to spleen, F5 targets to both spleen and lungs.

(103) Luciferase-RNA (20 μg) diluted in 1×PBS (A) or undiluted in water (B and C) was complexed with F4 liposomes diluted in 1×PBS (B) or undiluted in water (A and C) with an F4:RNA ratio of 1.2:2. The final PBS concentrations of all complexes were set to 1×PBS. BALB/c mice (n=5) were then injected intravenously with A, B or C and luciferase activities in spleens of mice were quantified via in vivo imaging (Mean+SD); see FIG. 15.

(104) As a standard mixing protocol, both liposomes and RNA are diluted in PBS (1×PBS final conc.) and then mixed at equal volumes. Predilution of only RNA is as good as standard protocol. All other protocols lacking predilution of RNA in PBS yielded poorer results. Presence of ions in RNA solution prior to complexation is preferred for achieving good results

(105) Luciferase-RNA (20 μg) precomplexed with 0.125 or 1 mM CaCl.sub.2 or without precomplexation was mixed via standard protocol with F4 liposomes with an F4:RNA ratio of 1.2:2. BALB/c mice (n=5) were injected intravenously with these formulations and luciferase activities in vivo were quantified from spleens of mice (Mean+SD); see FIG. 16.

(106) Precondensation of RNA with 1 mM CaCl.sub.2 when PBS is used as a buffer increases the luciferase signal 3-fold (Higher concentrations of CaCl.sub.2 in the presence of PBS leads to large particles-aggregates). Precondensation of RNA with Ca.sup.2+ helps to increase the luciferase signal.

(107) Luciferase-RNA (20 μg) or F4 liposomes diluted in 1×PBS or 154 mM NaCl were mixed with an F4:RNA ratio of 1.2:2. BALB/c mice (n=5) were injected intravenously with these formulations and luciferase activities in vivo were quantified from spleens of mice (Mean+SD); see FIG. 17.

(108) Using standard mixing protocol, replacement of PBS with isoosmolar NaCl worked as good as PBS.

(109) Luciferase-RNA (20 μg) precomplexed with 1-4 mM CaCl.sub.2 was mixed using standard protocol with F4 liposomes with an F4:RNA ratio of 1.2:2 using 154 mM NaCl instead of 1×PBS as dilution buffer. BALB/c mice (n=5) were injected intravenously with these formulations and luciferase activities in vivo were quantified from spleens of mice (Mean+SD); see FIG. 18.

(110) When PBS is replaced with NaCl, 2 mM CaCl.sub.2 can be used leading to 4.5-fold increase (higher concentrations of CaCl.sub.2 do not further increase the signal).

(111) Luciferase-RNA (5 μg) was incubated in 25 or 50% mouse serum for 30 min. and then electroporated into human monocyte derived immature DCs. Luciferase activity was assessed 18 h later via standard in vitro luciferase assay (Mean+SD); see FIG. 19A. Luciferase-RNA (20 μg) was complexed via standard protocol with F4 liposomes with an F4:RNA ratio of 1.2:2 and then incubated in the presence or absence of 50% mouse serum for 30 min.

(112) BALB/c mice (n=5) were injected intravenously with these formulations and luciferase activities in vivo were quantified from spleens of mice (Mean+SD); see FIG. 19B.

(113) Naked RNA is degraded in the presence of serum. Complexation of RNA with F4 liposomes protect it from RNase mediated degradation in serum.

(114) BALB/c mice (n=3) were injected intravenously with Cy5-RNA (40 μg) complexed with F4 liposomes labeled with Rhodamine (F4-rho) (1.2:2; Liposome:RNA). Uptake of Cy5-RNA or F4-rho by cell populations in spleen was assessed by flow cytometry 1 hour after lipoplex injection; see FIG. 20.

(115) As professional antigen presenting cells (APCs), splenic DCs and macrophages efficiently internalized the liposome encapsulated RNA and the liposome itself while B and T cells hardly internalized neither the liposome encapsulated RNA nor the liposome itself. Thus, RNA lipoplexes are selectively internalized by splenic APCs

(116) C57BL/6 mice (n=3) were injected with HA-RNA (40 μg) complexed with F4 (1.2:2; Liposome:RNA), F4 alone or PBS (as control); see FIG. 21. (A) Maturation status of dendritic cells (revealed by upregulation of CD86 and CD40) in spleen was determined by flow cytometry 24 hours after treatments (Mean+SD). (B) Serum concentrations of IFNa and TNFa were assessed via ELISA 6 and 24 hours after treatments (Mean+SD).

(117) As revealed by upregulation of activation markers (CD86, CD40) on DCs, RNA-F4 lipoplexes actived splenic DCs while liposome alone did not. Interestingly, although RNA-F4 lipoplexes were detected in 5-10% of splenic DCs in a previous experiment, all DCs were activated in spleen implying for the existence of an inflammatory milieu in spleen upon delivery. In all animals injected with RNA-lipoplexes, we could detect a high amount of IFNa in blood 6 h (also after 24 h although in much lower quantities). We could also detect TNFa but at very moderate levels in all animals injected with RNA-lipoplexes (only after 6 h). The secretion of cytokines is specific to RNA-lipoplexes as neither the PBS nor the liposome alone did not lead to any significant cytokine secretion (baseline). Thus, RNA lipoplexes activate splenic DCs leading to systemic inflammation

(118) C57BL/6 mice (n=5) were immunized intravenously with SIINFEKL-RNA (20 or 40 μg) complexed with F4 liposomes at different liposome:RNA ratios on days 0, 3, 8 and 15; see FIG. 22. (A) The frequencies of antigen specific CD8.sup.+ T cells were determined via SIINFEKL-MHC tetramer staining 5 days after the last immunization (Day 20) (Mean+SD). (B) Memory recall responses were assessed via SIINFEKL-MHC tetramer staining on Day 62 after another injection of F4-RNA lipoplexes on Day 57 (Mean+SD).

(119) High order of antigen-specific T cell immunity could be generated after repetitive immunization with F4 lipoplexes (A). 6 weeks after the last immunization (d57), a boost lipoplex injection was able to expand CD8 T cell memory formed in the former injections (B). F4 (1.2:1) complexes formed aggregates while F4 (1.2:2) complexes were clear. Preferred is F4 (1.2:2) with 40 μg RNA. Thus, strong T cell effector and memory responses can be generated with RNA-lipoplexes

(120) On days 0, 3 and 8, C57BL/6 mice (n=3) received three intravenous immunizations of SIINFEKL-RNA (40 μg) complexed with F4 liposomes with an F4:RNA ratio of 1.2:2 or left untreated. On day 14, 2×10.sup.5 B16-OVA tumor cells were injected s.c. into the flanks. Kaplan-Meier survival curves are shown in FIG. 23.

(121) Complete protection was achieved with RNA lipoplex administration in the prophylactic B16-OVA model.

(122) 2×10.sup.5B16-OVA tumor cells were inoculated s.c. into the flanks of C57/Bl6 mice (n=10, d0). At day 10 (tumor diameter 2-3 mm), mice received seven intravenous immunizations of SIINFEKL-RNA (40 μg) complexed with F4 or F12 liposomes with an F4:RNA ratio of 1.2:2 (on days 10, 13, 17, 24, 31, 38, 45). Liposomes alone without SIINFEKL-RNA were used as control treatment. Individual tumor growth and Kaplan-Meier survival curves are shown in FIGS. 24 and 25, respectively.

(123) In a therapeutic model, significantly delayed tumor growth for F4+RNA or F12+RNA groups was detected. Shrinkage of tumors after three immunizations were observed for both groups.

(124) BALB/c mice (n=3) were injected intravenously with Luciferase-RNA (20 μg) complexed with different amounts of F5 liposomes to yield F5:RNA ratios of 4.8:1, 2.4:1, 1.2:1, 1.2:2, 1.2:4. Luciferase activities in vivo and ex vivo were assessed via in vivo imaging 6 hours after lipoplex injection and representative mice and organ sets are shown in FIG. 26. FIG. 27 shows the distribution of total luciferase signal among organs derived from the experiment depicted in FIG. 26.

(125) F5 (DOTMA:Chol) goes to lungs at the ratio of F5:RNA of 4.8:1, to primarily lungs but also to spleen at the ratio of F5:RNA (2.4:1), to primarily spleen but also to lungs at the ratio of F5:RNA (1.2:1) and to exclusively to spleen at ratios of F5:RNA (1.2:2, 1.2:4). Neutral and anionic lipoplexes target more specifically to spleen whereas cationic lipoplexes primarily target lung (wrt to protein expression). No expression in liver was detected.

Example 12: Clinical Formulation of Lipoplexes

(126) The formulation following the previously established protocol consists of two steps, namely the preformulation of a given RNA by using isotonic sodium chloride solution as diluent and the lipoplex formation by adding a defined amount of liposomes. For preformulation, first 4 ml sodium chloride (0.9% w/w in water) solution will be taken out of the NaCl vial by a syringe and added to the RNA. Then, 400 μL of liposomes (2.8 mg/mL total lipid in water) will be taken out of the liposome vial and injected using a cannula (inner diameter of 0.9 mm) into the solution of RNA and sodium chloride. The obtained RNA lipoplex formulation (5.5 ml) can be administered either, by direct parenteral injection of the desired dose as well as after preparation of an intravenous infusion. To this end, from the RNA lipoplex formulation, 5.0 mL will be taken and diluted to an infusion bag containing 50 ml of isotonic sodium chloride solution. By this protocol, lipoplex formulations with particle sizes of about 300 to 500 nm are obtained in a robust and reproducible manner; see FIG. 28.

(127) Materials and components which may be used are as follows:

(128) Components:

(129) RNA: 0.5 mg/ml in 10 mM HEPES and 0.1 mM EDTA Diluent: 0.9% NaCl Liposomes: 2.68 mM DOTMA, 1.34 mM DOPE, particle size (Z.sub.ave) 300-500 nm
Syringes: 5 mL syringes: (e.g. Omnifix, 5 mL, Luer Lock, B. Braun Melsungen AG (Melsungen, Germany) 1 mL syringe: Injekt-F Tuberculin, 1 mL, Luer Lock, B. Braun Melsungen AG (Melsungen, Germany)
Needles: 0.9×44 mm, 20 G 1½″, BD Microlance 3, Becton Dickinson S.A. (Fraga, Spain)

(130) The sizes of the RNA lipoplex particles produced according to the above procedure range from 300 nm to 500; see FIG. 29.

Example 13: Effect of Particle Size

(131) It is demonstrated, that the activity of the lipoplexes increases with increasing size. The size of the liposomes used for formation of lipoplexes affects also the size of the lipoplexes. Larger liposomes lead also to larger lipoplexes.

(132) The particle characteristics of RNA lipoplexes reconstituted using F4 (DOTMA/DOPE 50:50 mol/mol) and F12 (DOTMA/DOPE 66.7:33.3 mol/mol) were investigated realizing different sizes of precursor. For that, particle sizing of lipoplexes with extruded liposomes and non-extruded, 0.45 μm filtered liposomes was performed.

(133) TABLE-US-00001 TABLE 1 Sizes of liposomes used for lipoplex formation Formulation Size extruded Size not extruded F4 164 nm 582 nm F12 163 nm 637 nm

(134) Results for the lipoplexes are shown in FIG. 30. It is demonstrated that lipoplexes of different sizes can be produced by using precursors of different sizes.

(135) The results from FIGS. 31 and 32 indicate that the bigger the liposomes the bigger the formed lipoplexes in these experiment the higher the observed luciferase signal.

Example 14: Sodium Chloride Buffer

(136) Several experiments have shown that addition of PBS buffer to the RNA prior to addition of liposomes, leads to an increase of the activity of the lipoplexes. Here it is demonstrated, that instead of PBS, normal saline solution (0.9% eg. 150 mM NaCl) can be used for RNA condensation. Such NaCl solution is available as approved medicinal drug product, which facilitates logistics and handling for the lipoplex-IMP. It is further demonstrated, that also concentrated solutions of NaCl and PBS can be used for RNA condensing, resulting in equivalent activity of the later formed lipoplexes. Furthermore detailed size measurements are shown, where differently concentrated NaCl solutions were added to RNA prior to lipoplex formation. In general, lipoplex size increases with decreasing concentration of the added NaCl solution; see FIG. 35. As increasing size is correlated to increasing activity (see Example 13), addition of the normal saline, and not the concentrated saline is considered to yield higher activity.

(137) To test the influence of pre-formulation of RNA using common buffers prior to the assembling, the particle size of lipoplexes at a charge ratio 1:2 were determined after treatment of the RNA with different concentrated PBS buffers or sodium chloride solutions; see FIGS. 33 and 34.

(138) The prior mixing protocol, where both liposomes and RNA are treated in PBS (lx PBS final conc.) and then mixed at equal volumes, can be replaced by a simpler mixing with normal sodium chloride solution (0.9%), which is commercially available as an approved medicinal drug product. As mixing protocol for the lipoplex-IMP, RNA is preformulated with isotonic saline solution and then mixed with the liposomes in water.

(139) The results suggest that the monovalent ion can be added at different concentrations in order to obtain the same final ionic strength in the lypoplex formulation without significantly affecting the lipoplex properties.

Example 15: Liposome/RNA Charge Ratio

(140) The charge ratio (ratio cationic lipid to nucleotide) of 1.3 to 2 is suitable regarding the physicochemical characteristics and the biological activity. At this ratio, a higher fraction of RNA is assumed to be included in the lipoplexes as for the ratio 1:2.

(141) The colloidal stability, the particle characteristics and the Luciferase activity of lipoplexes of non-extruded liposomes were further investigated. Lipoplexes were assembled in isotonic saline solution with liposome/RNA charge ratios between 1:2 and 1.9:2, see FIGS. 36 and 37. For lipoplexes, at a charge ratio of 1.7:2 the particle sizes significantly increased over time. In accordance with lipoplexes of extruded liposomes, lipoplexes with a charge ratio between 1:2 and 1.6:2 are invariant to minor changes in excess RNA and show particle sizes in the 350 to 480 nm range with PI values of less than 0.3.

(142) As demonstrated in FIG. 38, liposome/RNA charge ratios between 1.1:2 and 1.6:2 result in good activity in the spleen.

(143) All ratios deliver RNA exclusively to spleen without significant changes in performance between the different lipid/RNA ratio.