Small liposomes for delivery of immunogen encoding RNA
Nucleic acid immunization is achieved by delivering RNA encapsulated within a liposome. The RNA encodes an immunogen of interest, and the liposome has a diameter in the range of 60-180 nm, and ideally in the range 80-160 nm. Thus the invention provides a liposome having a lipid bilayer encapsulating an aqueous core, wherein: (i) the lipid bilayer has a diameter in the range of 60-180 nm; and (ii) the aqueous core includes a RNA which encodes an immunogen. These liposomes are suitable for in vivo delivery of the RNA to a vertebrate cell and so they are useful as components in pharmaceutical compositions for immunizing subjects against various diseases.
1. A pharmaceutical composition comprising a population of liposomes and a population of immunogen-encoding RNA molecules,
wherein the average diameter of the liposomes in the population is about 100 nm, with a polydispersity index of less than about 0.1, wherein said population of liposomes comprise: (i) 20%-80% (molar percent) cationic lipid or lipids; (ii) a zwitterionic lipid; and (iii) 20%-80% (molar percent) cholesterol; wherein at least half of the population of RNA molecules are encapsulated in liposomes, wherein said population of RNA molecules are self-replicating RNA molecules that encode (a) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA and (b) an immunogen, and wherein said population of RNA molecules are not packaged in virion particles.
2. The liposome of claim 1 , wherein the RNA is 5000-25000 nucleotides long.
3. The liposome of claim 1 , wherein the self-replicating RNA is a (+)-stranded RNA.
4. The liposome of claim 1 , wherein the self-replicating RNA has two open reading frames, the first of which encodes an alphavirus replicase and the second of which encodes the immunogen.
5. The liposome of claim 2 , wherein the self-replicating RNA is 9000-12000 nucleotides long.
6. The liposome of claim 1 , wherein the immunogen can elicit an immune response in vivo against a bacterium, a virus, a fungus or a parasite.
7. A pharmaceutical composition comprising a liposome of claim 1 .
8. The pharmaceutical composition of claim 7 , wherein said population of liposomes comprise 35%-50% (molar percent) cholesterol.
9. The liposome of claim 1 , wherein said liposome comprises DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane), DSPC (1,2-Diastearoyl-sn-glycero-3-phosphocholine), DPyPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), a cholesterol, a PEGylated lipid, or a combination thereof.
10. The pharmaceutical composition of claim 1 , wherein said population of liposomes comprise 35%-50% (molar percent) cholesterol.
11. The pharmaceutical composition of claim 1 , wherein said population of liposomes comprise 30%-70% (molar percent) cationic lipid or lipids.
12. A method for raising a protective immune response in a vertebrate, comprising the step of administering to the vertebrate an effective amount of the pharmaceutical composition of claim 1 .