Modified EC7 cells having low toxicity to viral production payloads
Recombinant cells and methods therefor are contemplated that allow for rapid and high titer production of recombinant viruses, and especially replication deficient Ad5 virus. In some preferred aspects, the host cell is modified to produce an inhibitor that reduces or eliminates the expression of a therapeutic protein encoded in the virus, while in other aspects, the virus includes a gene that directly or indirectly reduces or eliminates the expression of a therapeutic protein encoded in the virus. Most preferably, shRNA encoded by the host cell will reduce or suppress expression of a payload gene encoded in the recombinant virus.
1. A method of producing two preparations of recombinant therapeutic adenoviruses, the method comprising:
providing EC7 cells, wherein each of the EC7 cells express from a recombinant nucleic acid, a chimeric protein comprising a lambda repressor portion and a nuclear location sequence (NLS), wherein the NLS sequence is downstream of the lambda repressor;
providing a first plurality of recombinant adenoviruses comprising a genome with a first recombinant sequence portion that encodes a first cargo sequence and an operator sequence and a promotor sequence operably linked to the cargo sequence;
producing a first preparation of recombinant therapeutic adenovirus by (a) transfecting a first plurality of the EC7 cells with the genomes of the first plurality of recombinant adenoviruses, and (b) culturing the first plurality of transfected EC7 cells until they produce a first viral titer of at least 10 9 viral particles/mL;
providing a second plurality of recombinant adenoviruses comprising a genome with a second recombinant sequence portion that encodes a second cargo sequence and an operator sequence, and a promotor sequence operably linked to the cargo sequence:
producing a second preparation of recombinant therapeutic adenovirus by (a) transfecting a second plurality of the EC7 cells with the genomes of the second plurality of recombinant adenovirus, and (b) culturing the second plurality of transfected EC7 cells until they produce a second viral titer of at least 10 9 viral particles/mL;
wherein there is 20% variation or less between the time necessary to culture the first preparation to a titer of at least 10 9 viral particles/mL and the time necessary to culture the second preparation to a titer of at least 10 9 viral particles/mL.
2. The method of claim 1 , wherein the first and the second plurality of EC7 cells further expresses CXADR from a recombinant nucleic acid.
3. The method of claim 1 , wherein the first plurality and/or the second plurality of recombinant adenovirus are E2b-deleted adenovirus.
4. The method of claim 1 , wherein the first plurality and/or the second plurality of recombinant adenovirus genomes further comprises a viral payload gene that encodes at least one of a cytokine, a chimeric protein, a tumor associated antigen, and a neoepitope.
5. The method of claim 1 , wherein the first and the second viral titer is at least 10 10 viral particles/mL.
6. The method of claim 1 , wherein the first and the second viral titer is reached within a time period having a variability of equal or less than 10% between the first and the second EC7 cells.
7. The method of claim 1 , wherein the first cargo sequence and the second cargo sequence are the same.
8. The method of claim 1 , wherein the first cargo sequence and the second cargo sequence are different.