3.2 Two Baculovirus system: Two-Bac
The next generation baculovirus constructs design was driven by two key objectives: (1) the increase in expression stability of BV constructs, and (2) the reduction in number of baculoviruses for process simplification (Figure 3). In 2008, Chen proposed a strategy to improve the baculovirus stability with the introduction of a synthetic intron in the coding region of Rep (Bac-Intron-Rep: BacIn Rep) and Cap (Bac-Intron-Cap: BacIn Cap) sequences[33]. The synthetic intron consisted of a splice acceptor and a donor sequence flanking polh promoter sequence, which, when placed in a specific position upstream to Rep52 or VP2 stably expressed Rep and Cap over the extended passage numbers. The placement of a synthetic intron in BacIn Rep facilitated the independent expression of Rep78 and Rep52 from two different mRNA transcripts with improved stability. Similarly, in BacIn Cap, the synthetic intron drove independent expression of VP1 and VP2/3 from two different mRNA transcripts, respectively offering not only stability but also desired stoichiometry of all three VP proteins. The system was further simplified by combining both expression cassettes onto a single baculovirus: BacIn RepCap now requiring only two baculovirus co-infection for AAV production (Figure 2A).
Addressing the stability issue of Bac-Rep construct, in 2009, Smith et al. reported a different strategy leveraging ribosomal leaky scanning mechanism for stable expression of Rep proteins[34]. Here, therep gene sequence was altered which consisted of (1) a modified weak translational initiation codon for Rep78-CUG and (2) Modification of nine downstream AUG codons without altering the functionality of Rep proteins. This resulted in a non-duplication of Rep78 and Rep52 sequences and circumvented the root cause of the genetic instability of the original Bac-Rep construct. These modifications resulted in a weak Kozak sequence for Rep78, and subsequent leaky scanning of Rep78 and Rep52 codons. As a result, the stable expression of both Rep78 and Rep52 in a desired proportion was achieved from a single mRNA transcript. Next, to reduce the number of baculovirus vectors from three to two, the original VP expression cassette was combined and arranged in transcriptionally opposite direction with reference to Bac-Rep cassette (Figure 2A). This combination resulted in Bac-Rep2CapX recombinant baculovirus now requiring dual baculovirus co-infection for AAV production.