HR system allows better genetic stability of GOI over serial passages
Next, we decided to investigate the stability of recombinant baculovirus generated by HR and Tn7 systems. A baculovirus vector carrying a GFP reporter gene flanked by the AAV2 ITRs was produced either using the HR- or the Tn7-based method. Two types of baculovirus “rep/cap” were generated, one allowing the expression of AAV8 capsid and the other one the expression of AAVanc80 capsid variant. Of note, AAVanc80 has been recently identified as an in silico reconstructed ancestral AAV capsid with high transduction efficiency in-vivo (Zinn et al., 2015). We selected five clones per construct, and we performed side by side comparison of the baculovirus stability over 10 serials passages in Sf9 cells. To that purpose, the copy number of AAV ITR (itr) or Rep sequence (rep), and the copy number of the baculovirus DNA polymerase gene (bac) was determined by qPCR at passages P1, P4, P7 and P10. HR system showed extended genomic stability of the insert with a consistent ratio bac/itr or bac/rep up to ten passages (Fig. 3a – left ). Whatever the insert, the baculoviruses generated with Tn7 system are more heterogeneous between clones and has the tendency to loose the insert after 7 passages (Fig. 3a - right ). Finally, the expression of the Rep and Cap proteins required for the rAAV vector production was assessed by Western blotting. Positive signals for VP1, VP2, VP3 proteins were observed up to passage P10 for each HR-derived clone (Fig. 3b - top ), while a drastic drop in capsid protein expression was observed from passage P8 for the Tn7-derived clones (Fig. 3b - bottom ). Altogether, these observations suggest that HR system robustly prevents the loss of inserted cassettes upon passages, which is a major advantage for large-scale manufacturing of recombinant baculovirus, not only for rAAV production but also for vaccine or recombinant protein production.