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].