Reverse genetics using non-endogenous pol I promoters
View Patent ↗Expression of a transgene is driven in a host cell using a pol I promoter which is not endogenous to an organism from the same taxonomic order from which the host cell is derived.
1. A method for producing a recombinant virus, comprising a step of growing a canine kidney host cell comprising at least one expression construct encoding a viral RNA molecule, wherein expression of the viral RNA molecule from the construct is controlled by a human pol I promoter under conditions where the viral RNA molecule is expressed in order to produce virus.
2. A method of preparing a virus, comprising steps of: (i) producing a recombinant virus by the method of claim 1 ; (ii) infecting a culture host with the virus obtained in step (i); (iii) culturing the host from step (ii) to produce further virus; and (iv) purifying virus obtained in step (iii).
3. A method for preparing a vaccine, comprising steps of (a) preparing virus by the method of claim 2 and (b) preparing vaccine from the virus.
4. The method of claim 3 , wherein step (b) comprises formulating the vaccine with an adjuvant.
5. The method of claim 4 , wherein the adjuvant comprises a terpenoid.
6. The method of claim 5 , wherein the terpenoid is squalene or saponin.
7. The method of claim 4 , wherein the adjuvant comprises an oil-in-water emulsion.
8. The method of claim 7 , wherein the oil-in-water emulsion adjuvant comprises squalene, polysorbate 80, and sorbitan trioleate.
9. The method of claim 8 , wherein the squalene, polysorbate 80, and sorbitan trioleate are in a relative weight ratio to each other of about 4.3:0.5:0.48.
10. The method of claim 1 wherein the canine kidney cell is an MDCK cell.
11. The method of claim 10 wherein the MDCK cell is cell line MDCK 33016 (DSM ACC2219).
12. The method of claim 1 , wherein the canine kidney cell includes at least one bi-directional expression construct.
13. The method of claim 1 , wherein the expression construct is an expression vector or a linear expression construct.
14. The method of claim 1 , wherein the human pol I promoter comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
15. The method of claim 1 , wherein the virus is a segmented virus.
16. The method of claim 1 , wherein the virus is a non-segmented virus.
17. The method of claim 1 , wherein the virus is a negative-strand RNA virus.
18. The method of claim 17 , wherein the virus is influenza virus.
19. A canine kidney host cell for producing a virus comprising at least one expression construct encoding a viral RNA molecule, wherein expression of the viral RNA molecule from the construct is controlled by a human pol I promoter.
20. A canine kidney cell for producing a virus having at least one endogenous pol I promoter which control(s) expression of endogenous rRNA and at least one non-endogenous human pol I promoter which control(s) expression of a viral RNA or the complement thereof.