Novel approaches to render stable producer cell lines viable for the commercial manufacturing of rAAV-based gene therapy vectors

Abstract

Background Recombinant Adeno-associated virus (rAAV) based vectors recently emerged as very promising candidates for viral gene therapy due to a large toolbox available including twelve different AAV serotypes, natural isolates, designer capsids and library technologies [2]. Furthermore, rAAV vectors have favourable properties such as non-pathogenicity of AAV, low B-/T-cell immunogenicity against transgenes delivered and long-term transgene expression from a non-integrating vector [5,9]. Promising data from clinical trials using rAAVbased vectors for the treatment of e.g. haemophilia or retinal diseases as well as the recent approval of the first gene therapy drug in the European Union, Glybera to treat lipoprotein lipase deficiency, emphasise the potential of rAAV vectors for gene therapy approaches in a wide variety of indications [8,7,15]. Thereby, the demand for robust and cost-effective manufacturing of those vectors for market supply rose steadily. Standard production systems comprise transient transfectionand/or infection-based approaches using mammalian cells [3], or insect cells [16]. However, high production costs combined with considerable regulatory effort and safety concerns gave rise to the development of producer cell lines enabling stable rAAV production [3]. AAVs are parvoviruses whose productive infection is depending on the presence of helper viruses like e.g. adenovirus (AdV). Their single-stranded DNA genome carries two genes. The rep gene encodes proteins responsible for site-specific integration, viral genome replication as well as packging. The cap gene is translated into three structural proteins building the capsid shelf. Furthermore, cap encodes a protein required for capsid assembly (AAP or assembly-activating protein) that has been described recently [13]. The AAV genes are flanked by inverted terminal repeat (ITR) sequences constituting the replication, integration and packaging signal. In a stable producer cell line with integral helper functions, all required genetic elements are stably integrated into the genome of the host cell as independent expression constructs: the recombinant vector implying a transgene flanked by AAV ITRs, the AAV genes rep and cap required for replication and encapsidation, as well as adenoviral helper function delivered by sequences encoding genes E1a, E1b, E2a, E4orf6 and viral associated (VA) I/II RNA [9]. In a timely regulated fashion, viral proteins are expressed and the AAV genome is replicated and encapsidated. As some of the gene products arising during rAAV production are toxic, an inducible expression of the gene products is indispensable for generation of stable production cells. The aim of the underlying study is to provide all tools necessary to generate a stable and versatile producer cell line In order to circumvent the problems triggered by toxic proteins inevitably arising during rAAV formation, one objective of the project is to establish stable producer cells where rAAV production can be induced by temperature shift at the final production scale. To begin with, we first performed some general feasability studies to investigate whether the generation of stable and inducible producer cell lines using proprietary constructs is a viable approach. For this purpose, experiments for rAAV manufacturing based on a transient packaging approach were conducted. Infection of rep, cap and rAAV vector plasmid transfected cells with wildtype Adenovirus was compared with co-tranfection of the cells with additional plasmids carrying the Adenoviral * Correspondence: Verena.Emmerling@rentschler.de Rentschler Biotechnologie GmbH, Erwin-Rentschler-Strase 21, 88471 Laupheim, Germany Full list of author information is available at the end of the article Emmerling et al. BMC Proceedings 2013, 7(Suppl 6):P12 http://www.biomedcentral.com/1753-6561/7/S6/P12