METHODS OF MAKING LIPID FORMULATIONS WITH VIRAL IMMUNOGENS
RNA encoding an immunogen is delivered in a liposome for the purposes of immunisation. The liposome includes lipids which have a pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine. These liposomes can have essentially neutral surface charge at physiological pH and are effective for immunisation.
1 . A method of obtaining a formulation, the formulation comprising:
RNA molecules comprising a sequence that encodes an immunogen, wherein the immunogen comprises a respiratory syncytial virus (RSV) immunogen, an Epstein-Barr virus (EBV) immunogen, a cytomegalovirus (CMV) immunogen, a coronavirus spike polypeptide immunogen, an influenza virus A immunogen, a Varicella zoster virus (VZV) immunogen, or a flavivirus immunogen; and
lipids comprising a tertiary amine cationic lipid, a polyethylene glycol-conjugated (PEG-conjugated) lipid, and cholesterol; and
wherein the lipids encapsulate at least half of the RNA molecules;
the method comprising a first mixing of the lipids and the RNA molecules under conditions wherein the lipids encapsulate the at least half of the RNA molecules, thereby obtaining the formulation.
2 . The method of claim 1 , wherein the PEG-conjugated lipid comprises a PEG that has a molecular weight of 2000 Daltons.
3 . The method of claim 1 , wherein the PEG-conjugated lipid comprises 1,2-dimyristoyl-sn-glycero-2-phosphoethanolamine-N-[methoxy(polyethylene glycol)].
4 . The method of claim 1 , wherein the RNA molecules further comprise a 3′ poly(adenosine monophosphate) tail.
5 . The method of claim 4 , wherein the RNA molecules further comprise a 7′ methylguanosine and a 5′ first ribonucleotide, and wherein the 7′ methylguanosine is linked 5′-to-5′ to the 5′ first ribonucleotide.
6 . The method of claim 5 , wherein the 5′ first ribonucleotide comprises a 2′-methylated ribose.
7 . The method of claim 6 , wherein the lipids comprise from 35 mole % to 50 mole % of the cholesterol.
8 . The method of claim 6 , wherein the lipids comprise from 40 mole % to 60 mole % of the tertiary amine cationic lipid.
9 . The method of claim 6 , wherein the tertiary amine cationic lipid has a pKa from 5.0 to 7.6.
10 . The method of claim 6 , wherein the tertiary amine cationic lipid has a pKa from 5.6 to 6.8.
11 . The method of claim 6 , wherein the tertiary amine cationic lipid has a neutral charge at physiological pHs.
12 . The method of claim 1 , wherein the RNA molecules further comprise a sequence that encodes a replicase, and wherein the RNA molecules are self-replicating RNA molecules.
13 . The method of claim 6 further comprising a second mixing of the lipids and ethanol prior to the first mixing, thereby obtaining an ethanolic lipid mixture.
14 . The method of claim 13 further comprising a third mixing of the RNA molecules and an aqueous buffer prior to the first mixing.
15 . The method of claim 14 , wherein the aqueous buffer is a citrate buffer.
16 . The method of claim 15 , wherein the ethanolic lipid mixture is heated prior to the first mixing.
17 . The method of claim 6 , wherein the lipids further comprise 1,2-distearoyl-sn-glycero-3-phosphocholine.
18 . The method of claim 17 , wherein the PEG-conjugated lipid comprises a PEG that has a molecular weight of 2000 Daltons.
19 . The method of claim 17 , wherein the lipids comprise from 35 mole % to 50 mole % of the cholesterol.
20 . The method of claim 17 , wherein the tertiary amine cationic lipid has a pKa from 5.0 to 7.6.
21 . The method of claim 17 , wherein the tertiary amine cationic lipid has a pKa from 5.6 to 6.8.
22 . The method of claim 17 , wherein the tertiary amine cationic lipid has a neutral charge at physiological pHs.
23 . The method of claim 20 , wherein the lipids comprise from 40 mole % to 60 mole % of the tertiary amine cationic lipid.
24 . The method of claim 21 , wherein the lipids comprise from 40 mole % to 60 mole % of the tertiary amine cationic lipid.
25 . The method of claim 22 , wherein the lipids comprise from 40 mole % to 60 mole % of the tertiary amine cationic lipid.
26 . The method of claim 17 further comprising a second mixing of the lipids and ethanol prior to the first mixing, thereby obtaining an ethanolic lipid mixture.
27 . The method of claim 26 further comprising a third mixing of the RNA molecules and an aqueous buffer prior to the first mixing.
28 . The method of claim 27 , wherein the aqueous buffer is a citrate buffer.
29 . The method of claim 28 , wherein the ethanolic lipid mixture is heated prior to the first mixing.
30 . The method of claim 21 , wherein the nitrogen in the lipids is in a ratio to the phosphate in the RNA molecules from 4:1 to 16:1.