3.1. Three Baculovirus (BV) System: Three-Bac
The Three-Bac system achieved the production of the functional
recombinant AAV2 particles via co-infection of Spodoptera frugiperda
(Sf9) cells with three recombinant AcMNPV vectors [22]. In this
particular study, these baculovirus vectors were identified as Bac-VP
(Cap), Bac-Rep, and Bac-ITR, which harbored the AAV2 cap gene,
AAV2 rep gene, and ITR flanked, for example, by gfp gene
sequence cassettes as a transgene, respectively (Figure 2A). In general,
the insect cell promoters in combination with cellular machinery are
crucial to drive the production of foreign proteins in insect cells.
During the development of the Three-Bac system, the main challenges
faced were related to the optimization of molecular expression cassettes
to achieve proportionate expression of capsid subunits and replicase
proteins[29]. The main challenge with replicase protein expression
was due to the colinear ORFs in AAV genome wherein Rep52 and its p19
promoter sequences are in-frame with the entire Rep78 sequence[28].
This arrangement also led to the duplication of sequences and any
modifications such as insertion of insect promoter in the Rep52 sequence
to facilitate transcription in insect cell will result in an altered
sequence of entire Rep78 and its functional characteristics. Therefore,
Urabe et al[22]. separately arranged Rep78 and Rep52 expression
cassettes in transcriptionally opposite orientation (Bac-Rep) under
different insect promoters to achieve their desired expression level
(Figure 2A). On the other hand, the challenge with the capsid proteins
was achieving stoichiometric expression of three VP proteins in a
prototypic ratio of 1:1:10 for VP1:VP2: VP3. Sufficient expression and
incorporation of VP1 in AAV capsid is critical for efficient
transduction[30], [31]. During AAV production in mammalian
cells, the intron splicing and leaky scanning of translation initiation
codon by ribosomal complex results in differential expression of three
VP proteins in the desired proportion which was not observed in insect
cell[22]. As a result, the Bac-VP expression cassette was designed
with a modified cap sequence consisting of a weak VP1 translation
initiation codon-ACG in combination with a complex of nine nucleotide
sequence to achieve the stochastic ratio of VP proteins. The resulting
AAV2 particles demonstrated biophysical and functional characteristics
identical to that of mammalian-cells produced AAV2[22].
An important finding by Urabe et al[22]. was that unlike mammalian
cells, AAV production in insect cells via BEVS does not require an
additional helper virus such as Adenovirus or Herpes simplex virus
(HSV), and the necessary helper function is provided by the baculovirus
in a fashion possibly distinct yet functionally comparable to that of
Adenovirus or HSV. It can also be inferred that the insect cell
adequately provided the components and cellular machinery necessary for
AAV protein production, capsid assembly, genome replication, and its
packaging.
The original Three-Bac system, although extremely promising and
flexible, found minimal application due to the operational complexity
from the bioprocessing standpoint and the instability of all
three-helper baculoviruses coding rep , cap , and transgene
cassette. Continuously generating, characterizing, and validating three
baculoviruses seed stocks during AAV production runs was a significant
undertaking to assure process consistency and poses a significant
burden. Moreover, Kohlbrenner et al. reported the passage dependent loss
of AAV protein expression in these BVs, notably the loss of Rep
proteins. The loss of expression of Rep proteins, which are essential
for genome replication and its encapsidation, resulted in an overall
reduced yield of packaged and functional AAV particles[32].
Palindromic orientation with Rep78 and consequent excision of the entire
Rep 52 sequence was believed to be the reason for Bac-Rep instability
(Figure 2A). When isolated onto different baculoviruses, the stability
of Rep78 and Rep52 was restored over the extended BV passage, however,
requires quadruple BV co-infection further enhancing operational
complexity[32].