3.4 Low VP1 Expression and Functional AAV yields in Insect
Cells
In the mammalian cells, AAV’s natural host, the combination of mRNA
splicing of p40 promoter-driven transcript and leaky ribosomal scanning
of VP2’s weak translation of initiation codon-ACG result into the
expression of three VP subunits in a standard ratio of 1:1:10 for
VP1:VP2:VP3 from a mRNA transcript. The VP1 carries the sequence of
PLA2-like enzymatic domain and nuclear localization
signal, both of which are critical due to their postulated role in AAV
transduction[30], [40]. Therefore, the expression of VP1 in an
appropriate proportion and its subsequent incorporation in the AAV
capsid is crucial to generate functional viral particles. Due to the
inherent difference of splicing mechanism in insect and mammalian cells,
in the original Three-Bac system, authentic start codon AUG of VP1 was
mutated to weak ACG codon facilitating leaky scanning via insect cell
ribosomal machinery achieving expression of all three VP proteins in a
proportion comparable to mammalian cells[22]. The Three-Bac though
originally successful for AAV2, was found to be non-versatile for other
serotypes such as AAV5 and AAV8[32]. AAV5 being the evolutionary
most distant serotype[41], is of important clinical significance due
to its relatively better immuno-privileged characteristics compared to
other serotypes that are closely related to human origin serotype AAV2.
The AAV5, when produced in insect cell, exhibited no or lesser
transduction efficiency, which was found to be associated with
insufficient VP1 expression[32], [39], [42]. The deficient
VP1 expression was linked to the AAV5 VP1 sequence and the leaky
scanning efficiency in insect cells. The efficiency of ribosomal
scanning for recognizing AUG as a translation initiation codon relies on
Kozak sequence: a sequence context surrounding AUG, importantly
ACCAUGG, where G at +4 position is crucial (A of AUG being +1) in
case of weak initiation codon such as ACG[43], [44]. In contrast
to AAV2, the G of +4 position is U, in the case of AAV5, which is
believed to be responsible for deficient expression and subsequent
lesser incorporation of VP1 in AAV5 capsid[42]. Urabe et al.
addressed this challenge by domain swapping where 86 amino acid long
AAV5 VP1 sequence at specific positions was substituted with that of
AAV2 VP1 sequence resulting in AAV5 chimeric capsid with infectivity
comparable to that of AAV2[42]. Similarly, Kohlbrenner et al.
performed the domain swapping of an entire AAV5 and AAV8 VP1 sequences
with that of AAV2 VP1, resulting in increased VP1 proportion and
PLA2 activity with restoration of transduction
efficiency[32]. In One-Bac2.0, the second generation One-Bac, the
mutated VP1 initiation codon (ACG) was replaced with an authentic VP1
strong codon (AUG), which in combination with synthetic intron placement
strategy as reported earlier by Chen[33], demonstrated stronger
expression of all three VP proteins from two mRNA transcripts and
restored original composition of VP1 and AAV5 functionality. The RBE
negative version of this cell line (∆RBE) also significantly reduced the
cross packaging of rep/cap sequences and hence the proportion of
replication competent viral particles confirming the role of RBE as a
packaging signal[45]. Overall, these modifications improved the
final quality of the AAV5 serotypes produced using One-Bac2.0. Further
improved version was the third generation system:One-Bac3.0 where the
canonical VP1 initiation codon AUG was restored and was located in
serotype-specific strategically modified Kozak context sequence to
achieve optimal expression level of all three VP proteins[46]. Here,
the strong AUG codon was surrounded by a weak Kozak sequence, which led
to suboptimal translation initiation efficiency of 40%-45% compared to
optimal mammalian Kozak sequence. As a result, reduced yet sufficient
VP1 expression was achieved, and concomitant leaky scanning of following
VP2/VP3 start codons restored their optimal expression ratio with
respect to VP1. AAV5 and AAV9 serotypes produced using this system
demonstrated in-vivo transduction efficiency better than or comparable
to those produced in mammalian cells. Coupled with the ∆RBE strategy,
this system produced vectors with only marginal encapsidation of foreign
DNA sequences as reported with next-generation sequencing
analysis[46].
Recently, in the Two-Bac system, the selection of optimal translation
initiation codon and associated downstream nucleotide sequence which
exhibited optimal stoichiometric expression of all three VP proteins of
AAV5 with improved functionality of the resultant vector was reported
[47].