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 method for producing a recombinant adeno-associated virus (AAV) vector, the method comprising introducing into a eukaryotic host cell a first phagemid particle comprising a recombinant AAV transgene cassette, a second phagemid particle comprising rep and cap genes, and a third phagemid particle comprising an adenohelper gene, and allowing the host cell to produce recombinant AAV vector.
2. The method of claim 1 , wherein the eukaryotic host cell is a mammalian cell.
3. A recombinant phagemid particle comprising rAAV, rep-cap and adenohelper DNA sequences.
4. A recombinant phagemid particle comprising a transgene expression cassette, wherein the one or more transgene expression cassette comprises left and right Inverted Terminal Repeat sequences (ITRs) of an adeno-associated virus (AAV) serotype, wherein the phagemid particle lacks bacteriophage structural genes required for the formation, packaging or extrusion of the particle from a prokaryotic host, wherein the phagemid particle lacks structural genes which encode capsid proteins.
5. The recombinant phagemid particle of claim 4 , wherein the recombinant phagemid particle comprises a packaging signal.
6. The recombinant phagemid particle of claim 5 , wherein the packaging signal comprises an origin of replication.
7. The recombinant phagemid particle of claim 6 , wherein the origin of replication is an F1 ori or a pUC ori.
8. The recombinant phagemid particle of claim 4 , wherein the recombinant phagemid particle comprises a DNA sequence that favors targeted integration into a host genome.
9. The recombinant phagemid particle of claim 4 , wherein the transgene expression cassette comprises one or more functional elements required for expression of the transgene in a target cell selected from: a promoter, nucleic acid for encoding a poly A tail attachable to the expressed agent, left and right Inverted Terminal Repeat sequences (ITRs).
10. The recombinant phagemid particle of claim 4 , wherein the recombinant phagemid particle comprises a pIII capsid minor coat protein that is configured to display a cell-targeting ligand for enabling delivery of the particle to a target cell.
11. The recombinant phagemid particle of claim 4 , wherein the recombinant phagemid particle comprises a pVIII capsid major coat protein that is configured to display a foreign peptide thereon.
12. The recombinant phagemid particle of claim 4 , wherein the recombinant phagemid particle is combined with a cationic polymer to form a complex having a net positive charge.
13. The method of claim 1 , wherein the first phagemid particle, the second phagemid particle, and the third phagemid particle each lacks bacteriophage structural genes required for the formation, packaging or extrusion of the particle from a prokaryotic host, and lacks structural genes which encode the capsid proteins.
14. The recombinant phagemid particle of claim 3 , wherein the recombinant phagemid particle lacks bacteriophage structural genes required for the formation, packaging or extrusion of the particle from a prokaryotic host, wherein the phagemid particle lacks structural genes which encode the capsid proteins.