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

Lionel Galibert,Bérangère Bertin,Delphine Bonnin,Adrien Savy,Otto-Wilhelm Merten,Isidore Mushimiyimana,Monique M Van Oers,Yohann Dickx,Yi Li

doi:10.1371/journal.pone.0207414

Lionel Galibert, Bérangère Bertin + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0207414

Copy DOI

Abstract

The ability to produce large quantities of recombinant Adeno-Associated Virus (rAAV) vectors is an important factor for the development of gene therapy-based medicine. The baculovirus/insect cell expression system is one of the major systems for large scale rAAV production. So far, most technological developments concerned the optimization of the AAV rep and cap genes in order to be expressed correctly in a heterologous system. However, the effect of the baculovirus infection on the production of rAAV has not been examined in detail. In this study we show that the baculoviral cathepsin (v-CATH) protease is active on several (but not all) rAAV serotypes, leading to a partial degradation of VP1/VP2 proteins. Subsequently, we identified the principal v-CATH cleavage site in the rAAV8 capsid proteins and demonstrated that the cleavage is highly specific. The proteolytic degradation of VP1/VP2 AAV capsid proteins reduces the infectivity of rAAV vectors but can be prevented by the use of a baculovirus vector with a deletion of the chiA/v-cath locus or by addition of the E64 protease inhibitor during production. Moreover, the codon optimization of AAV cap performed for several serotypes and originally aimed at the removal of potential alternative initiation codons, resulted in incorporation of additional forms of truncated VP1 into the rAAV capsids.

Highlights

Gene delivery vectors derived from Adeno-Associated Virus (AAV) are widely used for development of treatments against a range of rare genetic diseases
Titers of recombinant Adeno-Associated Virus (rAAV) obtained in the bulk were in the same range for the rAAV produced with the unmodified bacmid system (WT) as for rAAV produced with the Δ-chiA/vcath baculovirus (ΔCC) [2.5 x 1011 viral genomes.mL-1 ± 1.3 x 1010 versus 1.9 x 1011 vg.mL-1 ± 3.5 x 1010, as averaged over 3 replicates for both wild type bacmid (WT) and ΔCC]
Western blot analysis of the same samples, using the B1 antibody (Progen), which recognizes the C-terminal part of the three AAV capsid proteins [21] identified the major extra protein band as well as the two minor supplementary bands observed with the WT baculovirus (Fig 1B)

Summary

Introduction

Gene delivery vectors derived from Adeno-Associated Virus (AAV) are widely used for development of treatments against a range of rare genetic diseases. In 2002, the first rAAV vector based on the baculovirus-insect cell expression system was made available [1]. Three recombinant baculoviruses were used, one encoding the rAAV-genome, a second carrying the AAV capsid (cap) genes and a third one encoding the AAV large and short replicase (rep) genes. The second generation rAAV production system combined rep and cap in a single recombinant baculovirus [2].

Methods

Results

Conclusion