Methods and kits for the improved fermentative production of a recombinant virus
AssigneeTakeda Pharmaceutical Company Limited
InventorsJuan Antonio Hernandez Bort, Carolin Isabel Kahlig, Lucia Micutkova, Barbara Krauss, Johannes Grillari
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Abstract
The present invention relates to methods and kits for the improved fermentative production of recombinant adeno-associated virus (AAV), adenovirus, lentivirus or retrovirus such as gammaretrovirus in HEK293 cells. Further, uses for increasing the fermentative production of said viruses in HEK293 cells are described.
Claims39
A method for the fermentative production of an adeno-associated virus (AAV), an adenovirus, a lentivirus or a retrovirus comprising the steps of:
- Step 1:
- culturing the rAAV, the adenovirus, the lentivirus or the retrovirus in a HEK293 cell in a culture medium comprising an amount of an interferon (IFN) inhibitor over an incubation period;
- and
- Step 2:
- recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture.
A method for the fermentative production of an adeno-associated virus (AAV), a lentivirus or a retrovirus comprising the steps of:
- Step 1:
- performing a transcriptomic analysis on HEK293 cells transfected with an rAAV, an adenovirus, a lentivirus or a retrovirus and selecting the HEK293 cell if the transcription of at least one of the following genes is enriched compared to a negative control:
- (i) TNFA_signaling_via_NFKB;
- (ii) Interferon_Gamma_Response;
- (iii) Interferon_Alpha_Response;
- (iv) TGF_beta_Signaling;
- and/or
- (v) IL6_JAK_STAT3_Signaling,
- preferably if the transcription of
- (1) interferon_gamma response;
- and/or
- (2) interferon_alpha_respone
- is enriched;
- Step 2:
- culturing the rAAV, the adenovirus, the lentivirus or the retrovirus in the selected HEK293 cell in a culture medium comprising an amount of an interferon (IFN) inhibitor over an incubation period;
- and
- Step 3:
- recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture.
Use of an IFN-inhibitor for increasing the yield of rAAV, adenovirus, Lentivirus or retrovirus in a culture of HEK293 cells transfected or infected with the rAAV, the adenovirus, the lentivirus or the retrovirus.
A kit for use in cell culture comprising
- (a) HEK293 cells;
- and
- (b) an IFN inhibitor selected from the group consisting of Ruxolitinib, Tofacitinib, Baricitinib, Delgocitinib, Emapalumab and Fontolizumab and any structural analog thereof having IFN inhibitor activity, preferably the IFN inhibitor is Ruxolitinib and any structural analog thereof having at least 50% of the inhibitory activity for the JAK family kinases in the IFN signaling pathway.
Dependencies are inferred from the claim text.