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].