Particles comprising single stranded RNA and double stranded RNA for immunomodulation

1. A homogeneous chimeric particle comprising a complex;

wherein the complex consists of at least one cationic agent, a single stranded RNA (ssRNA) and a double stranded RNA (dsRNA); wherein the at least one cationic agent comprises protamine; wherein the complex is formed by the addition of the at least one cationic agent to premixed ssRNA and dsRNA; and wherein the dsRNA comprises two RNA molecules fully or partially hybridized together.

2. The particle of claim 1 , wherein the particle has a diameter in the range of from about 50 nm to about 1000 nm.

3. The particle of claim 1 or 2 , wherein the at least one cationic agent encloses said RNA.

4. The particle of claim 1 , wherein the at least one cationic agent comprises a polycationic compound.

5. The particle of claim 1 , wherein the at least one cationic agent comprises at least one agent selected from the group consisting of an RNA-complexing lipid, an RNA complexing polymer, and an RNA-complexing peptide or protein.

6. The particle of claim 1 , wherein the at least one cationic agent comprises at least one agent selected from the group consisting of polyethyleneimine, a poly-L-lysine, a poly-L-arginine or a histone.

7. The particle of claim 1 , wherein the at least one cationic agent consists of protamine.

8. The particle of claim 1 , wherein the protamine: RNA (ssRNA+dsRNA) weight ratio is from 16:1 to 1:2, preferably from 4:1 to 1:2.

9. The particle of claim 1 , wherein the ssRNA contains at least one U nucleotide and/or at least one G nucleotide.

10. The particle of claim 1 , wherein the ssRNA is an oligonucleotide of from 6 to 100 nucleotides or an mRNA of from 50 to 10,000 nucleotides.

11. The particle of claim 1 , wherein the ssRNA is an oligonucleotide having the sequence according to SEQ ID NO: 1.

12. The particle of claim 1 , wherein the strands of the dsRNA are in average from 6 to 8000 nucleotides in length.

13. The particle of claim 1 , wherein the dsRNA is polyinosinic-polycytidylic acid (poly(I:C)).

14. The particle of claim 1 for use as an immunostimulant.

15. A pharmaceutical composition comprising the particle of claim 1 and optionally one or more pharmaceutically acceptable carriers, diluents and/or excipients.

16. The pharmaceutical composition of claim 15 which further comprises at least one adjuvant.

17. A method for stimulating the immune system of a subject comprising administering to the subject the pharmaceutical composition of claim 15 or 16 .

18. The method of claim 17 , wherein the pharmaceutical composition is co-administered with at least one antigen.

19. A method for the preparation of homogeneous chimeric particles comprising the steps of:

(a) providing an aqueous solution of single stranded RNA (ssRNA);

(b) providing an aqueous solution of double stranded RNA (dsRNA);

(c) providing an aqueous solution of protamine; and

(d) combining the solutions obtained in steps (a) and (b) and mixing it with the solution obtained in (c); and

wherein the chimeric particles comprise a complex consisting of protamine, ssRNA and dsRNA.

20. A method for the preparation of homogeneous chimeric particles comprising the steps of:

(a) providing an aqueous solution of single stranded RNA (ssRNA) at less than 5 mg/ml using a solution containing 0 to 125 mM electrolytes;

(b) providing an aqueous solution of double stranded RNA (dsRNA) at less than 5 mg/ml using a solution containing 0 to 125 mM electrolytes;

(c) providing an aqueous solution of protamine at less than 5 mg/ml using a solution containing 0 to 125 mM electrolytes; and

(d) combining the solutions obtained in steps (a) and (b) and adding the solution obtained in (c); and

wherein the chimeric particles comprise a complex consisting of protamine, ssRNA and dsRNA.

21. The method of claim 19 or 20 , wherein step (a) and/or step (b) comprises resuspending dried RNA in an aqueous solution containing 0 to 125 mM electrolytes.

22. The method of claim 19 or 20 , wherein step (c) comprises diluting an aqueous isotonic stock solution containing 1000 to 5000 heparin-neutralizing units of protamine per ml with an aqueous solution containing 0 to 125 mM electrolytes.

23. The method of claim 19 or 20 comprising the steps of:

(a) providing an aqueous solution of ssRNA at less than 5 mg/ml by resuspending dried ssRNA in pure water;

(b) providing an aqueous solution of dsRNA at less than 5 mg/ml by resuspending dried dsRNA in pure water;

(c) providing an aqueous solution of protamine at less than 5 mg/ml by diluting an aqueous isotonic stock solution containing 5000 heparin-neutralizing units of protamine per ml with pure water; and

(d) combining the solutions obtained in steps (a) and (b) and adding the solution obtained in (c).