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.