Phagemid vector
The invention provides hybrid and recombinant phagemid vectors for expressing a transgene in a target cell transduced with the vector. A recombinant phagemid particle comprises at least one transgene expression cassette which encodes an agent which exerts a biological effect on the target cell, characterised in that the phagemid particle comprises a genome which lacks at least 50% of its bacteriophage genome. The invention extends to the use of such phagemid expression systems as a research tool, and for the delivery of transgenes in a variety of gene therapy applications, DNA and/or peptide vaccine delivery and imaging techniques. The invention extends to in vitro, in vivo or in situ methods for producing viral vectors, such as recombinant adeno-associated viruses (rAAV) or lentivirus vectors (rLV), and to genetic constructs used in such methods.
1. A recombinant phagemid particle comprising bacteriophage capsid proteins and a transgene expression cassette, wherein the phagemid particle lacks structural genes that encode the bacteriophage capsid proteins.
2. The recombinant phagemid particle of claim 1 , wherein the transgene expression cassette is a viral transgene expression cassette, optionally wherein the transgene expression cassette is an adeno-associated virus (AAV) transgene expression cassette.
3. The recombinant phagemid particle of claim 2 , wherein the transgene expression cassette is flanked by Inverted Terminal Repeat sequences (ITRs).
4. The recombinant phagemid particle of claim 1 , further comprising a bacteriophage origin of replication.
5. The recombinant phagemid particle of claim 4 , wherein the bacteriophage origin of replication is an F1 ori.
6. The recombinant phagemid particle of claim 1 , further comprising a bacterial origin of replication.
7. The recombinant phagemid particle of claim 6 , wherein the bacterial origin of replication is a pUC ori.
8. The recombinant phagemid particle of claim 1 , wherein the recombinant phagemid particle is combined with a cationic polymer to form a complex having a net positive charge.
9. The recombinant phagemid particle of claim 8 , wherein the cationic polymer is selected from a group consisting of: chitosan; poly-D-lysine (PDL); diethylaminoethyl (DEAE); diethylaminoethyl-dextran (DEAE.DEX); polyethyleneimine (PEI); polybrene; protamine sulphate; and a cationic lipid.
10. The recombinant phagemid particle of claim 1 , wherein the recombinant phagemid particle comprises a DNA sequence that favors targeted integration into a host genome.
11. The recombinant phagemid particle of claim 1 , wherein the transgene expression cassette encodes an agent which exerts a biological effect on a target cell.
12. The recombinant phagemid particle of claim 11 , wherein the agent is a protein agent or an RNA agent.
13. The recombinant phagemid particle of claim 1 , wherein the recombinant phagemid particle comprises a capsid coat protein that is configured to display a cell-targeting ligand for enabling delivery of the particle to the target cell.
14. The recombinant phagemid particle of claim 13 , wherein the capsid coat protein is a pIII minor coat protein or a pVIII capsid major coat protein.
15. The recombinant phagemid particle of claim 1 , wherein the recombinant phagemid particle comprises a capsid coat protein that is configured to display a foreign peptide thereon.
16. The recombinant phagemid particle of claim 15 , wherein the capsid coat protein is a pIII minor coat protein or a pVIII capsid major coat protein.
17. The recombinant phagemid particle of claim 1 , wherein the bacteriophage capsid proteins are M13 capsid proteins.
18. A pharmaceutical composition comprising the recombinant phagemid particle of claim 1 .
19. A pharmaceutical composition comprising the recombinant phagemid particle of claim 11 .
20. A method of performing gene therapy on a subject, the method comprising administering to the subject the pharmaceutical composition of claim 19 .