Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system

Abstract

The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce wtAAV2 in the baculovirus/Sf9 cell system hypothesizing that the wtAAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce wtAAV2 in Baculovirus/Sf9, we found that wtAAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system leads to the expression of Rep78 that finally excises wtAAV2 genome from the baculovirus genome during the earliest phases of baculovirus stock production. Via p5 promoter expression kinetics and strand specific RNA-Seq analysis of wtAAV2, rAAV and Rep2/Cap2 cassettes in the baculovirus context we could demonstrate that wtAAV2 native promoters, p5, p19 and p40 are all active in the context of the baculovirus system and lead to the expression of different proteins and peptides. In addition, this study has proven that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle.

Origins of truncated supplementary capsid proteins in rAAV8 vectors produced with the baculovirus system

“…By comparing several rAAV serotypes we were able to demonstrate that another supplementary band (slightly smaller than the VP1 protein) was not a degradation product of the VP1 protein but originated from internal translation initiation in codon optimized cap sequences. Interestingly, we have previously shown that rep gene codon optimization could lead to supplementary transcription events compared to a non-optimized rep gene construct [ 31 ]. Thus codon optimization of AAV genes in the context of baculovirus expression system should be carefully checked and compared to non-optimized genes.…”

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

“…In contrast, in the Two-Bac system, Smith et al [35] A more recent modification utilized the weak initiation codon for Rep78, which enabled partial exon skipping and subsequent expression of downstream Rep52 from a single expression cassette and reportedly enhanced Bac-Rep stability for at least five passages. [41] In the Mono-Bac, [37] the rep-cap expression cassette reported in Two-Bac [35] was integrated into the egt (ecdysteroid UDP- Rep proteins was also critical to avoid Rep-induced excision of AAV expression cassette [42] and its integration stability in baculovirus genome as this can ultimately affect the stability of Mono-Bac BEV and overall production titer of AAV vectors.…”

“…The very late phase expression of Rep proteins, specifically after baculovirus DNA replication, minimized Rep‐induced excision of the rep‐cap expression cassette from the baculovirus genome, resulting in sustained expression stability. Furthermore, this late phase expression of Rep proteins was also critical to avoid Rep‐induced excision of AAV expression cassette [ 42 ] and its integration stability in baculovirus genome as this can ultimately affect the stability of Mono‐Bac BEV and overall production titer of AAV vectors.…”

Monobac System–A Single Baculovirus for the Production of rAAV

“…Although expression platforms are usually trade secrets, most rAAV production systems use a variety of co-infecting AAV helper viruses. The adenoviral system has been developed and improved in terms of safety by using only essential sets of “early” adenovirus genes (e.g., E1, E2, E4, and VA RNA I and II), expressed from plasmid, without the lytic “late” genes [ 15 , 16 , 17 , 18 ]. In this set-up, the E1 gene is generally supplied by a producer cell line (HEK293, PER.C6, Hela-E1).…”

“…This system uses three baculoviruses to collocate into the Sf9 cell the AAV rep and cap genes along with the rAAV transgene [ 17 ]. A significant problem with this early work was the inability of the Sf9 cell to recognize the splicing signal used by AAV [ 18 ]. This meant that the regulation of VP1-VP2-VP3 ratios was solely achieved through a ribosome scanning mechanism, linked to the use of non-ATG initiation codons.…”