## 3 Using IC-BEVS for AAV Production  
### Development of AAV Production Systems  
Insect cell lines serve as effective production platforms for viral vectors, and several research groups have developed the Baculovirus (BV) structures to facilitate the formation, DNA replication, and subsequent packaging of AAV capsids within Sf9 cells [63]. In the initial approach using baculoviruses to generate rAAV vectors, in 2002, [PERSON] and [PERSON] et al. devised a production strategy based on three distinct recombinant BV-infected Sf9 cells, known as the Three-Bac system (Figure 3A) [64]. The first BV carries \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Name** & **Baculovirus** & **Recombinant Method** & **Advantages/Disadvantages** & **References** \\ \hline \multirow{4}{*}{Bac-to-Bac} & Circular; & In vitro; & Allow for the rapid isolation of the & \\  & Contain the _lacZ_ gene & Transpositional & recombinant baculoviruses; long-term & \\  & & & and work-intensive screening of & \\  & & & recombinant baculoviruses & \\  & & & Eliminate additional screening of & \\  & & & In vitro; & positive clones; improve the efficiency & \\  & & & Transpositional & of transposition; reduce the cost and & \\  & & & recombination & time required for high-throughput & \\  & & & baculovirus production & \\  & & Circular; & In vitro; & Suitable for virus-like particle design; & \\  & & Contain multiple & Transpositional & recombinant baculoviruses are & [60] \\  & & expression frames & recombination & not stable & \\  & & Circular; & In vivo, & Faster baculovirus production with & \\  & & Delete & In vivo, & maximum expression of secreted & \\  & & & Homologous & proteins without cumbersome & \\  & & genes & & plaque purification & \\  & & & In vivo, & Pure viral clones can be obtained & \\  & & gene with _SacB_ gene & homologous & without a plaque purification; & \\  & & recombination & cost-effective & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Development of insect baculovirus vector systems. the Rep78 and Rep52 genes, the second BV carries the Cap gene, and the third BV carries the ITR transgene. Rep78 and Rep52 are expressed in two separate boxes controlled by the baculovirus Immediate Early stage (AIE1) and Polyhedral protein (polh) promoters, respectively. The expression of the three VP proteins in a single box controlled by the polh promoter, along with the mutation of the VP1 translation initiation codon to ACG, enables the synthesis of VP2 and VP3, allowing the three VP proteins to be expressed in appropriate proportions without the need for alternative splicing. The Cap is co-induced by another copy of the polh promoter. Finally, the transgene is linked to a gene promoter active in human cells, flanked by an ITR, and inserted into a third baculovirus. Although AAV2 vectors have been successfully produced using this method, each virus requires co-infection of insect cells to form rAAV, which is impractical for long-term applications. Due to the genetic instability of BEV and the consequent loss of AAV production, as well as the inability to produce functional AAV vectors for other serotypes, this method has not been widely adopted [65]. The same authors later developed a BV infection process by replacing Rep52 of AAV1 with an analogue of AAV5 and the N-terminal portion of AAV2 VP1 with an analogue of AAV5, achieving a considerable yield (approximately four times) and increased infectivity [66]. Subsequent development of the IC-BEVS production system of AAV has led to the reduction in the number of vectors required to two, termed the Two-Bac system (Figure 3B). In 2009, [PERSON] and [PERSON] et al. reported the expression of Rep and Cap proteins of AAV by using leaky ribosome scanning (Figure 3(Ba)). The modified AAV 2 Rep gene is controlled by the transcription of the baculovirus polyhedron protein promoter. The bifunctional Rep mRNA transcript uses the CUG triplet embedded in the Kozak consensus sequence to guide the synthesis of Rep78 polypeptide.