3.3 One Baculovirus System: One-Bac1.0
The One-Bac system reported by Aslanidi et al. was a further adaptation
of the IC-BEVS platform for rAAV production[35]. This system
consists of two components: (1) A baculovirus-inducible stably
transformed packaging Sf9 cell line harboring the AAV2 rep andcap sequences under the transcriptional control of polh promoter
and (2) a single recombinant baculovirus carrying AAV2 ITR flanked
transgene/gene of interest sequences (Bac-ITR). The inducible cell line
offers the advantage of regulated yet amplified expression of AAV
proteins resulting in a higher yield of AAV, whereas the stable
packaging cell line ensures consistent expression levels requiring only
a single baculovirus infection.
The development of this system provided answers to two key questions,
(1) how to devise a regulation switch for inducible expression of AAV
proteins in insect cells? and (2) how to achieve sustained and higher
expression of incorporated Rep and Cap proteins in the desired
proportion? The initial unsuccessful attempt of generating stable cell
line suggested an absolute requirement of baculovirus genomic elements
in cis . One such element used in the final expression cassette
was a modified homologous region 2 (hr2-0.9), derived from A.
californica, which is inducible in the presence of immediate early
(IE-1) transcriptional trans -regulator, herein provided by
Bac-ITR baculovirus. Related to the second question, the optimal
expression level of Rep78/Rep52 is essential and has a direct effect on
the overall yield. The lower expression ratio of Rep78:Rep52 is favored
for better AAV yield[29]. In order to further improve the system,
the rep-binding element (RBE) was included in the rep and cap expression
cassette. This was based on the findings that the ITR and a complex of
p5 and rep-binding element (RBE), alone or in combination, upregulate
p19 promoter activity in mammalian cells[36], [37]. Further
studies reported the evidence of partial activity p19 promoter in the
insect cells[32], [38]. Combining these findings, the final
version of rep and cap expression cassettes to generate a
stable cell line consisted of hr2-0.9 and RBE regulatory elements
derived from baculovirus and AAV2, respectively (Figure 2A). As a
result, lower Rep78:Rep52 expression ratio and sustained higher
expression of rep and cap proteins (Figure 2B) were obtained with up to
ten times higher cell-specific yield of AAV2 when compared to the
original Three-Bac system.
The modular construct design of this One-Bac was further explored for
the production of AAV serotypes 1-12 [39]. The AAV production yield
for all the serotypes was either comparable or higher than the original
Three-Bac system. Notable though was the deficient VP1 expression and
consequent reduction in transduction efficiency of some serotypes,
specifically of AAV5.