Pharmaceutical composition consisting of RNA having alkali metal as counter ion and formulated with dications
A biologically active RNA-alkali metal-dication formulation, a pharmaceutical composition containing the complexes, and methods of producing the same. The formulation is particularly useful to introduce RNA and an attached cargo into cells allowing its biological intracellular activities: e.g. immunostimulation (immunomodulation), RNA interference or gene expression.
1. A composition comprising:
a complex of RNA and an alkali metal counter ion, the RNA having a sequence containing four consecutive G residues consisting of 5′-AGUGUUAUCUUGUAUGGGG-3′ (SEQ ID NO: 8); and
a dication-containing solution.
2. The composition of claim 1 wherein the dication is an alkaline earth metal.
3. The composition of claim 2 wherein the alkaline earth metal is calcium.
4. The composition of claim 1 wherein the alkali metal is sodium.
5. The composition of claim 1 wherein the RNA consists of unmodified nucleotide residues.
6. The composition of claim 1 wherein the RNA contains one or more modified nucleotide residues.
7. The composition of claim 1 wherein the RNA is linked to a chemical or biological cargo entity.
8. The composition of claim 1 together with a pharmaceutically acceptable excipient.
9. A method for stimulating a host immune response in a subject, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising a complex of RNA, the RNA having a sequence consisting of 5′-AGUGUUAUCUUGUAUGGGG-3′ (SEQ ID NO: 8), and a sodium counter ion in a calcium-containing solution.
10. The method of claim 9 wherein the subject is a mammal.
11. The method of claim 10 wherein the mammal is a human.
12. The method of claim 9 wherein the composition induces production of interferon-α in the subject.
13. A method for stimulation of the production of interferon-α in human immune cells in vitro, comprising administering to human immune cells in vitro an effective amount of a pharmaceutical composition comprising a complex of RNA, the RNA having a sequence consisting of 5′-AGUGUUAUCUUGUAUGGGG-3′ (SEQ ID NO: 8), and a sodium counter ion in a calcium-containing solution.
14. A method for the preparation of a composition comprising a complex of RNA with an alkali metal as counter ion in a dication-containing solution comprising the steps of:
(a) providing a RNA having a sequence consisting of 5′-AGUGUUAUCUUGUAUGGGG-3′ (SEQ ID NO: 8);
(b) next, preparing a RNA-alkali metal complex of the RNA having a sequence consisting of 5′-AGUGUUAUCUUGUAUGGGG-3′ (SEQ ID NO: 8) and an alkali metal counter ion; and
(c) then, adding a dication to the RNA-alkali metal complex.
15. The method of claim 14 wherein the step of preparing the RNA-alkali metal complex comprises ion exchange or precipitation.
16. The method of claim 15 wherein the step of preparing the RNA-alkali metal complex comprises precipitation of the RNA-alkali metal complex in a mixture of a sodium salt and an alcohol.
17. The method of claim 15 , wherein the step of preparing the RNA-alkali metal complex comprises precipitation of the RNA-alkali metal complex in a mixture of sodium chloride or sodium acetate, and ethanol.
18. The method of claim 14 , wherein the dication is calcium, and the composition comprising a complex of RNA with an alkali metal as counter ion in a dication-containing solution is an aqueous solution containing 0.2 to 20 mM calcium.
19. The method of claim 18 wherein an aqueous solution of 1 to 20 mM CaCl 2 to an aqueous solution of 1 to 10 mg/ml of the RNA-alkali metal complex the composition comprising a complex of RNA with an alkali metal as counter ion in a dication-containing solution has a final concentration of 0.2 to 20 mM Ca 2+ .
20. A composition comprising:
a complex of RNA and an alkali metal counter ion, the RNA having a sequence selected from the group consisting of:
(SEQ ID NO: 1)
5′-AGUGUUAUCUUGUAUGGGGGG-3′,
(SEQ ID NO: 8)
5′-AGUGUUAUCUUGUAUGGGG-3′,
(SEQ ID NO: 11)
5′-AGUGUUAUCUUGUAUUUUUUU-3′,
(SEQ ID NO: 12)
5′-AGUGUUAGGGGGGUCUUGUAU-3′,
(SEQ ID NO: 13)
5′-GGGGGGAGUGUUAUCUUGUAU-3′,
(SEQ ID NO: 14)
5′-AGUGUUAUCUUGUAUGGAAGG-3′,
and
(SEQ ID NO: 15)
5′-AUA UUC UUG UAU GGG GGG-3′;
and
a dication-containing solution.
21. The composition of claim 20 wherein the dication is an alkaline earth metal.
22. The composition of claim 21 wherein the alkaline earth metal is calcium.
23. The composition of claim 20 wherein the alkali metal is sodium.
24. The composition of claim 20 wherein the RNA is linked to a chemical or biological cargo entity.
25. A composition comprising:
a complex of RNA and an alkali metal counter ion, the RNA having a sequence selected from the group consisting of:
5′-AGUGUUAUCUUGUAUGGGGGG-3′ (SEQ ID NO: 1),
5′-AGUGUUAGGGGGGUCUUGUAU-3′ (SEQ ID NO: 12),
5′-GGGGGGAGUGUUAUCUUGUAU-3′ (SEQ ID NO: 13), and
5′-AUA UUC UUG UAU GGG GGG-3′ (SEQ ID NO: 15); and
a dication-containing solution.
26. The composition of claim 25 wherein the dication is an alkaline earth metal.
27. The composition of claim 25 wherein the alkaline earth metal is calcium.
28. The composition of claim 25 wherein the alkali metal is sodium.
29. The composition of claim 25 wherein the RNA is linked to a chemical or biological cargo entity.