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.