Bacteriophage-polymer hybrid
The invention provides a targeted bacteriophage-polymer complex comprising a recombinant targeted-bacteriophage and a cationic polymer. The complex has a net positive charge. The invention provides methods of preparing bacteriophages and complexes thereof, and to their uses for the delivery of transgenes in a variety of gene therapy applications.
1. A method of delivering a transgene to a mammalian cell, comprising:
administering to a mammalian cell a targeted bacteriophage-polymer complex comprising a recombinant negatively charged targeted-bacteriophage and a cationic polymer selected from the group consisting of poly-D-lysine (PDL), diethylaminoethyl-dextran (DEAE.DEX) and polyethyleneimine (PEI), wherein the recombinant bacteriophage comprises a nucleic acid sequence, which encodes a protein ligand that is capable of being expressed on the capsid coat of the recombinant bacteriophage, and which is specific for a protein expressed on a mammalian target cell or tissue, so as to enable targeted delivery thereto, wherein the complex has a net positive charge and a ζ-potential of at least 5 mV at physiological pH, and wherein the complex comprises a weight: weight ratio of about 100 ng to 400 ng polymer:1 μg phage.
2. The method of claim 1 , wherein the bacteriophage comprises a transgene, which exerts a therapeutic effect on a target cell, wherein the transgene comprises the Herpes simplex virus tyrosine kinase gene.
3. The method of claim 1 , wherein the ligand comprises the RGD4C ligand.
4. The method of claim 1 , wherein the bacteriophage is F1, Fd or M13.
5. The method of claim 1 , wherein the cationic polymer comprises DEAE.DEX.
6. The method of claim 1 , wherein the polymer comprises PDL.
7. The method of claim 1 , wherein the complex comprises a weight:weight ratio of about 130 ng-320 ng polymer:1 μg phage.
8. The method of claim 1 , wherein the bacteriophage-cationic polymer complex comprises calcium phosphate.
9. The method of claim 1 , wherein the ζ-potential of the bacteriophage-polymer complex is at least 10 mV or 15 mV at physiological pH.
10. A method of delivering a transgene to a mammalian cell, comprising:
administering to a mammalian cell an aggregate comprising a plurality of bacteriophage-polymer complexes comprising a recombinant negatively charged targeted-bacteriophage and a cationic polymer selected from the group consisting of poly D-lysine (PDL), diethylaminoethyl-dextran (DEAE.DEX) and polyethyleneimine (PEI), wherein the recombinant bacteriophage comprises a nucleic acid sequence, which encodes a protein ligand that is capable of being expressed on the capsid coat of the recombinant bacteriophage, and which is specific for a protein expressed on a mammalian target cell or tissue, so as to enable targeted delivery thereto, wherein the complex has a net positive charge and a ζ-potential of at least 5 mV at physiological pH, and wherein the complex comprises a weight:weight ratio of about 100 ng to 400 ng polymer:1 μg phage.
11. The method of claim 10 , wherein the mean particle size of the aggregate is between about 600 nm and 2500 nm, or between about 600 nm and 1500 nm, or between about 700 nm and 1200 nm.
12. A targeted bacteriophage-polymer complex comprising a recombinant negatively charged targeted-bacteriophage and a cationic polymer selected from the group consisting of poly-D-lysine (PDL), diethylaminoethyl-dextran (DEAE.DEX) and polyethyleneimine (PEI), wherein the recombinant bacteriophage comprises a nucleic acid sequence, which encodes a protein ligand that is capable of being expressed on the capsid coat of the recombinant bacteriophage, and which is specific for a protein expressed on a mammalian target cell or tissue, so as to enable targeted delivery thereto, wherein the complex has a net positive charge and a ζ-potential of at least 5 mV at physiological pH, and wherein the complex comprises a weight: weight ratio of about 100 ng to 400 ng polymer:1 μg phage.
13. A method of treating, preventing or ameliorating a disease in a subject using a gene therapy technique, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the targeted bacteriophage-polymer complex according to claim 12 , or an aggregate comprising a plurality of bacteriophage-polymer complexes according to claim 12 .
14. A method of producing a targeted bacteriophage-polymer complex of claim 12 , comprising:
contacting a recombinant targeted-bacteriophage with a cationic polymer to form a stable complex, which has a net positive charge.
15. The method of claim 14 , wherein the bacteriophage is incubated with the cationic polymer at a temperature of about 15-30° C., or about 18-25° C., or preferably about 20-23° C., for at least 5 mins, 10 mins or 15 mins.
16. A vaccine comprising the bacteriophage-polymer complex according to claim 12 , and optionally an adjuvant.
17. A genetic-molecular imaging technique comprising use of the bacteriophage-polymer complex according to claim 12 , wherein the technique comprises delivering the bacteriophage-polymer complex of claim 12 to a target cell, and imaging the target cell using a molecular imaging technique.
18. A pharmaceutical composition comprising the bacteriophage-polymer complex according to claim 12 , and a pharmaceutically acceptable vehicle.