Liposomes
Cationic liposomes with entrapped polynucleotide in the intravesicular space are described. The liposomes include cationic components such as cationic lipids such as DOTAP. Preferably the method of forming liposomes uses the dehydration-rehydration method in the presence of the polynucleotide. The polynucleotide preferably operatively encodes an antigen capable of eliciting a desired immune response, that is, is a gene vaccine.
1. A method to generate both a cell-based and humoral immune response to a target polypeptide in an animal, which method comprises administering subcutaneously or intramuscularly to the animal a composition comprising liposomes suspended in an aqueous liquid and a polynucleotide comprising a promoter operatively linked to a nucleotide sequence encoding said target polypeptide,
wherein the liposomes comprise phosphatidylcholine (PC), dioleoyl phosphatidyl ethanolamine (DOPE), and 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP), have diameters in the range 100 to 2000 nm and comprise a lipid bilayer and an aqueous intravesicular space,
wherein said polynucleotide is entrapped in the aqueous intravesicular space,
wherein said lipid bilayer includes said DOTAP in an amount such that the lipid bilayer has an overall cationic charge;
whereby said polynucleotide is delivered to and is expressed in target cells whereby an immune response including an IgG response and Th1 and Th2 responses to the target polypeptide result;
wherein said polynucleotide is administered in an amount sufficient to elicit said immune response.
2. The method of claim 1 , wherein said composition has been prepared by a process that comprises
mixing an aqueous suspension of empty liposomes with said polynucleotide to form a mixed suspension,
dehydrating the mixed suspension to form a dehydrated mixture, and
rehydrating the dehydrated mixture in an aqueous liquid to form liposomes which are dehydration-rehydration vesicles (DRVs) containing the polynucleotide in the intravesicular space.
3. The method of claim 2 , wherein said process further includes subjecting said polynucleotide-containing DRVs to microfluidization or extrusion.
4. The method of claim 1 , wherein the composition is administered intramuscularly.
5. A process for forming an aqueous suspension of liposomes having diameters in the range 100 to 2000 nm comprising the steps:
a) providing an aqueous suspension of small unilamellar vesicles formed from the liposome-forming agents phosphatidylcholine (PC), dioleoyl phosphatidyl ethanolamine (DOPE), and 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP), wherein said DOTAP is present in an amount whereby the small unilamellar vesicles have an overall cationic charge;
b) adding to the aqueous suspension of small unilamellar vesicles a nucleic acid including a promoter operatively linked to a nucleotide sequence encoding an immunogenic polypeptide to form a mixed suspension in which the weight ratio of liposome forming components making up the small unilamellar vesicles in step (a) to the nucleic acid added in step (b) is in the range (50 to 10000):1;
c) dehydrating the mixed suspension to form a dehydrated mixture;
d) rehydrating the dehydrated mixture to form an aqueous suspension of liposomes that are dehydration-rehydration vesicles (DRVs) containing said nucleic acid in an intravesicular space thereof; and
e) optionally subjecting the aqueous suspension of DRVs to microfluidisation whereby said aqueous suspension of liposomes is produced.
6. The process of claim 5 further comprising removing non-entrapped nucleic acid from the aqueous suspension of DRVs.
7. A composition for administration to an animal to induce a cell-based and humoral immune response to a polypeptide, which composition comprises
liposomes having diameters in the range 100 to 2000 nm and having lipid-bilayers surrounding aqueous intravesicular spaces and a polynucleotide comprising a promoter operatively linked to a nucleotide sequence encoding said target polypeptide,
which lipid-bilayers are formed from liposome forming components that comprise phosphatidylcholine (PC), dioleoyl phosphatidyl ethanolamine (DOPE), and 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP), and wherein said DOTAP is in an amount to confer an overall cationic charge on the liposomes, and
wherein the polynucleotide is entrapped in the aqueous intravesicular space.
8. The composition of claim 7 , wherein the liposomes are suspended in a pharmaceutically acceptable aqueous vehicle.
9. The composition of claim 7 , wherein the polypeptide is a viral polypeptide.
10. The composition of claim 9 , wherein the viral polypeptide is a polypeptide of hepatitis B, hepatitis C, influenza or human immunodeficiency virus.
11. The composition of claim 10 , wherein the viral polypeptide is hepatitis B surface antigen or haemagglutinin.