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

Adrien Savy,Otto-Wilhelm Merten,Yohann Dickx,Lionel Galibert,Adrien Léger,Minna U Kaikkonen,Jianming Qiu

doi:10.1371/journal.pone.0199866

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.

Highlights

Wild type adeno-associated virus has first been described as a contaminating virus in an adenovirus preparation [1] and was later developed as gene therapy vector because of its favorable features such as non-pathogenicity or its capacity to transduce dividing and nondividing cells/tissues [2]WtAAV2 is characterized by a 4,679 bases single-stranded DNA genome including Inverted Terminal Repeats (ITRs) [3] at both ends
PCR products were of 1 kb for the wtAAV2 cassette and of the expected size for the rAAV γ-SGC. These results indicated that the wtAAV2 genome was somehow excised from the bacmid backbone for all selected clones, but not the rAAV γ-SGC cassette used as control (Fig 1)
We describe in this study that wtAAV2 cannot be produced using the baculovirus/Sf9 cell system due to the unanticipated activity of p5 promoter leading to early expression of Rep78 that subsequently results in wtAAV2 genome excision from the baculovirus backbone via the combined action of ITR recognition and endonuclease activity

Summary

Introduction

Wild type adeno-associated virus (wtAAV) has first been described as a contaminating virus in an adenovirus preparation [1] and was later developed as gene therapy vector because of its favorable features such as non-pathogenicity or its capacity to transduce dividing and nondividing cells/tissues [2]WtAAV2 is characterized by a 4,679 bases single-stranded DNA genome including Inverted Terminal Repeats (ITRs) [3] at both ends. The cap gene codes for the 3 structural elements VP1, VP2 and VP3, which are overlapping sequences. The shorter mRNA encodes the two other structural elements, VP2 (72 kDa) which originates from a non-canonical start codon (ACG) and VP3 (62 kDa) which is translated from a downstream conventional start codon (AUG). These three structural proteins differ from each other at their N-terminus sequences. VPs assembly leads to an icosahedral capsid composed of 60 VP proteins displaying a VP1:VP2:VP3 ratio about 1:1:10 [8] This ratio reflects the relative efficiency of translation initiation of the three start codons and abundance of the two transcripts

Methods

Findings

Conclusion