## 3 Using IC-BEVS for AAV Production
### Development of AAV Production Systems
Some ribosome subunits fail to start translation at the non-standard start codon and scan the next AUG start codon to initiate the translation of Rep52 polypeptide. The AAV cap gene is controlled by the transcription of the p10 promoter of baculovirus. Three capsid proteins (VP1, VP2, and VP3) are translated from a single mRNA, and the sequences guiding the processing of 3\({}^{\prime}\)-mRNA come from simian virus 40 (SV40 pA) and herpes simplex virus thymidine kinase gene (HSV Uk pA) [67]. In 2008, [PERSON] et al. further modified the system to enable the expression of multiple proteins from a single Rep and Cap coding sequence by using artificial introns (Figure 3(Bb)). Polh promoters are placed in intron sequences to drive the expression of Rep and Cap short transcripts, resulting in the Rep52, VP2, AAP, and VP3 proteins. After splicing the internal polh promoter sequence, the Rep78 and VP1 proteins are translated from the longer transcript. Under this concept, Rep78/52 and VP1/2/3 are expressed by infecting one or two BVs, leading to an improved yield [68]. To overcome the drawback of low ratio with multi-BVs co-infection, the highly integrated One-Bac system has been developed, which was designed by Sf9 packaging cell lines dependent (Figure 3(Ca,b)) and Sf9 packaging cell lines independent (Figure 3(C,d)). Firstly, in 2009, [PERSON] et al. infected these cells with a single baculovirus vector carrying an ITR-flanked transgenic cassette, induced and amplified the expression of AAV proteins in an inducible Sf9/Rep-Cap packaging cell line (Figure 3(Ca)), resulting in a tenfold higher cell-specific yield of AAV2 vector particles compared to the Three-Bac system [69]. In 2014, [PERSON] et al. further utilized this One-Bac system to further expand the production of rAAVs from serotypes rAAV1 through rAAV12, and the highest rAAV yield can reach about 5 \(\times\) 10\({}^{5}\) vector genomes per cell (VG/cell) [70]. These researchers further developed the second generation One-Bac system (One-Bac 2.0) by the intron splicing-based strategy and removing REP-binding elements (RBE) from rep/cap expression constructs in the Sf9/Rep-Cap packaging cell lines, resulting enhanced VP1 expression of AAV5 and in less packaging of unwanted foreign DNA in rAAV particles of four serotypes (AAV1, AAV2, AAV5, and AAV8) [71; 72]. In 2019, [PERSON] et al. developed an improved One-Bac system composed of Sf9-GFP/Rep stable packaging cell line and BEV/Cap-(ITR-GOI) (Figure 3(C)). Obviously, this system is more versatile and flexible for the production of different serotypes of rAAVs [73]. Figure 3: Different BEVS for AAV production using Sf9 cells. (**A**) The Three-Bac systems. Three different recombinant baculoviruses were used to infect Sf9 cells, in which Bac-Rep carries Rep78 and Rep52, which were expressed in two separate boxes controlled by the baculovirus Immediate Early stage (\(\Delta\)IE1) and Polyhedral protein (polh) promoters, Bac-Cap carries Cap gene, and Bac-(ITR-GFP) carries CMV promoter, P10 promoter, GFP protein and polyadenylation site (pA) [(**B**)]; (**B**) The Two-Bac systems. (**a**) The Rep and Cap proteins are expressed by polh (PH) promoter and P10 promoter. The sequences guiding the processing of 3\({}^{\prime}\)-mRNA come from simian virus 40 (SV40 pA) and herpes simplex virus thymidine kinase gene (HSV tk pA) [(7)]. (**b**) Bac-Rep-Cap put P10 and PH promoters into intron sequences to drive the expression of Rep and Cap short transcripts to produce proteins [(68)]; (**C**) The One-Bac system designed by Sf9 packaging cell lines dependent (**a**,**b**) and Sf9 packaging cell lines independent (**c**,**d**). (**a**) Infection of these cells with a single baculovirus vector carrying an ITR-f flanked transgene cassette induces and amplifies the expression of AAV proteins [(69; 70; 71; 72)]. (**b**) Dual-function Bac-Cap-(ITR-GFP) carries both the AAV Cap gene and rAAV genome. Sf9-GFP/Rep packaging cell line harbors silent copies of the AAV2 Rep gene and constitutively expressed GFP reporter gene [(73)].