Cationic liposomes containing immune response generating moieties
AssigneeLipoxen Technologies Limited
InventorGregory GREGORIADIS
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Abstract
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.
Claims25
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 a phosphatidylcholine (PC), a phosphatidyl ethanolamine (PE), and a cation of the formula
- wherein each Y1 and Y2 is independently —O— or O—C(O)— wherein the carbonyl carbon is joined to R1 or R2 as the case may be;
- each R1 and R2 is independently an alkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms,
- each R3, R4 and R5 is independently hydrogen, alkyl of 1 to 8 carbon atoms, aryl or aralkyl of 6 to 11 carbon atoms, and wherein alternatively two of R3, R4 and R5 are combined with the positively charged nitrogen atom to form a cyclic structure having from 5 to 8 atoms, where, in addition to the positively charged nitrogen atoms, the atoms in the structure are carbon atoms and can include one oxygen, nitrogen or sulfur atom;
- n is 1 to 8;
- and
- X is an anion;
- wherein said liposomes have diameters in the range 100 to 2,000 nm and comprise a lipid bilayer and an aqueous intravesicular space,
- wherein said polynucleotide is entrapped in the aqueous intravesicular space of said liposomes,
- wherein said lipid bilayer includes said cation 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.
A method to generate an IgG1 response to a target polypeptide in an animal, which method comprises administering 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 consist essentially of a phosphatidylcholine, a phosphatidyl ethanolamine and a cationically charged liposome forming component;
- wherein said liposomes have diameters in the range 100 to 2,000 nm and comprise a lipid bilayer and an aqueous intravesicular space,
- wherein said polynucleotide is entrapped in the aqueous intravesicular space of said liposomes,
- wherein said lipid bilayer includes said cationically charged lipsome forming component 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 IgG1 response to the target polypeptide results;
- wherein said polynucleotide is administered in an amount sufficient to elicit said IgG1 response.
A process for forming an aqueous suspension of liposomes having diameters in the range 100 to 2,000 nm comprising the steps:
- a) providing an aqueous suspension of small unilamellar vesicles formed from the liposome-forming agents consisting essentially of phosphatidylcholine, a phosphatidyl ethanolamine and a cationically charged liposome forming component wherein said cationically charged liposome forming component 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 10,000):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.
A composition for administration to an animal to induce a IgG1 response to a target polypeptide, which composition comprises
- Liposomes having diameters in the range 100 to 2,000 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 consist essentially of phosphatidylcholine, a phosphatidyl ethanolamine and a cationically charged liposome forming component wherein said cationically charged liposome forming component wherein said cationically charged liposome forming component is present in an amount to confer an overall cationic charge on the liposomes, and
- wherein the polynucleotide is entrapped in the aqueous intravesicular space of said liposomes.
Dependencies are inferred from the claim text.