Delivery of RNA to trigger multiple immune pathways
RNA encoding an immunogen is co-delivered to non-immune cells as the site of delivery and also to immune cells which infiltrate the site of delivery. The responses of these two cell types to the same delivered RNA lead to two different effects, which interact to produce a strong immune response against the immunogen. The non-immune cells translate the RNA and express the immunogen. Infiltrating immune cells respond to the RNA by expressing type I interferons and pro-inflammatory cytokines which produce a local adjuvant effect which acts on the immunogen-expressing non-immune cells to upregulate major histocompatibility complex expression, thereby increasing presentation of the translated protein to T cells. The effects on the immune and non-immune cells can be achieved by a single delivery of a single RNA e.g., by a single injection.
1. A method of obtaining a composition, the composition comprising lipid particles and messenger ribonucleic acid (mRNA) molecules; the mRNA molecules comprising: (a) a 5′ cap nucleoside, (b) a first 5′ ribonucleoside, (c) a triphosphate bridge, and (d) a sequence that encodes a respiratory syncytial virus (RSV) surface fusion glycoprotein (F-protein) immunogen; the first 5′ ribonucleoside comprising a 2′-methylated ribose; the 5′ cap nucleoside being linked 5′-to-5′ to the first 5′ ribonucleoside by the triphosphate bridge; the lipid particles comprising lipids comprising: (a) a polyethylene glycol-ylated lipid, (b) cholesterol, (c) an anionic phospholipid or a zwitterionic phospholipid, and (d) a cationic lipid comprising a tertiary amine; and the lipid particles encapsulating at least half of the mRNA molecules;
the method comprising the steps of:
(i) mixing the lipids and ethanol, thereby obtaining an ethanolic lipid mixture;
(ii) mixing the mRNA molecules and an aqueous buffer, thereby obtaining an aqueous RNA mixture;
(iii) mixing the ethanolic lipid mixture and the aqueous RNA mixture, thereby obtaining an intermediate mixture; and
(iv) purifying the intermediate mixture, thereby obtaining the composition.
2. The method of claim 1 , the mRNA molecules comprising a modified nucleotide.
3. The method of claim 2 , the 5′ cap nucleoside being a 7-methylguanosine.
4. The method of claim 1 , the lipids comprising the zwitterionic phospholipid; and the zwitterionic phospholipid being 1,2-distearoyl-sn-glycero-3-phosphocholine.
5. The method of claim 2 , the lipids comprising the zwitterionic phospholipid; and the zwitterionic phospholipid being 1,2-distearoyl-sn-glycero-3-phosphocholine.
6. The method of claim 3 , the lipids comprising the zwitterionic phospholipid; and the zwitterionic phospholipid being 1,2-distearoyl-sn-glycero-3-phosphocholine.
7. The method of claim 1 , the mRNA molecules being self-replicating RNA.
8. The method of claim 1 , wherein in step (iv), the purifying comprises a tangential flow filtration.
9. The method of claim 2 , wherein in step (iv), the purifying comprises a tangential flow filtration.
10. The method of claim 5 , wherein in step (iv), the purifying comprises a tangential flow filtration.
11. The method of claim 6 , wherein in step (iv), the purifying comprises a tangential flow filtration.
12. The method of claim 1 , the ethanolic lipid mixture being heated prior to step (iii).
13. The method of claim 2 , the ethanolic lipid mixture being heated prior to step (iii).
14. The method of claim 5 , the ethanolic lipid mixture being heated prior to step (iii).
15. The method of claim 6 , the ethanolic lipid mixture being heated prior to step (iii).
16. The method of claim 10 , the ethanolic lipid mixture being heated prior to step (iii).
17. The method of claim 11 , the ethanolic lipid mixture being heated prior to step (iii).
18. The method of claim 1 , the aqueous buffer being a citrate buffer.
19. The method of claim 2 , the aqueous buffer being a citrate buffer.
20. The method of claim 11 , the aqueous buffer being a citrate buffer.
21. The method of claim 11 , the aqueous buffer being a citrate buffer.
22. The method of claim 1 , at least 80% of the lipid particles having a diameter in the range of 20-220 nm.
23. The method of claim 6 , at least 80% of the lipid particles having a diameter in the range of 20-220 nm.
24. The method of claim 5 , at least 80% of the lipid particles having a diameter in the range of 20-220 nm.
25. The method of claim 6 , at least 80% of the lipid particles having a diameter in the range of 20-220 nm.
26. The method of claim 24 , wherein in step (iv), the purifying comprises a tangential flow filtration.
27. The method of claim 25 , wherein in step (iv), the purifying comprises a tangential flow filtration.
28. The method of claim 8 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
29. The method of claim 9 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
30. The method of claim 10 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
31. The method of claim 11 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
32. The method of claim 20 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
33. The method of claim 21 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
34. The method of claim 26 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
35. The method of claim 27 , the tangential flow filtration comprising a membrane that has a 100 kDa pore size cutoff.
36. The method of claim 32 , the tangential flow filtration comprising a phosphate-buffered saline.
37. The method of claim 33 , the tangential flow filtration comprising a phosphate-buffered saline.
38. The method of claim 1 , the mixing of step (iii) being performed with a T-mixer.
39. The method of claim 2 , the mixing of step (iii) being performed with a T-mixer.
40. The method of claim 3 , the mixing of step (iii) being performed with a T-mixer.
41. The method of claim 5 , the mixing of step (iii) being performed with a T-mixer.
42. The method of claim 6 , the mixing of step (iii) being performed with a T-mixer.
43. The method of claim 10 , the mixing of step (iii) being performed with a T-mixer.
44. The method of claim 11 , the mixing of step (iii) being performed with a T-mixer.
45. The method of claim 20 , the mixing of step (iii) being performed with a T-mixer.
46. The method of claim 21 , the mixing of step (iii) being performed with a T-mixer.