Baculoviruses derived from HR or Tn7 systems allow the
generation of recombinant AAV viral vectors at similar production yields
We performed rAAV production using the co-infection strategy described
by Smith and collaborators with the recombinant baculoviruses derived
either from HR or Tn7 system (Smith et al., 2009). To this end, Sf9
cells were infected with a BEV ITR-GFP and a BEV rep/cap at a
multiplicity of infection (MOI) of 1 infectious unit each, as determined
by CSA assay. Insect cells were harvested 96 hours post-infection to
maximize production of rAAV using low MOI (Cecchini et al., 2011). We
first evaluated rAAV production yields by qPCR both at harvest in the
cleared lysate. Interestingly, baculovirus vectors derived from HR and
Tn7 systems yielded similar levels of rAAV particles whatever the capsid
variant (Fig. 4a ). The HR-derived BEV seems to lead to slightly
more empty particles, even if statistically not significant
(Fig. 4b ). This may be related to the absence of chiAand v-cath proteases, as suggested previously (Chen, 2017). After
purification, the infectivity of rAAV stocks was analyzed calculating
the ratio vector genomes/infectious particles. No difference in term of
infectivity was observed between vectors by using the infection center
assay (ICA) method (Fig. 4c ), which evaluate the capability of
rAAV particles to enter HeLaC32 cells and replicate in the nucleus in
the presence of Rep and Adenovirus (François et al., 2018). Altogether,
these observations suggested that the HR system is comparable to Tn7 to
produce rAAV in suspension Sf9 insect cells.