Volume 7 Supplement 6
Novel approaches to render stable producer cell lines viable for the commercial manufacturing of rAAV-based gene therapy vectors
© Emmerling et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
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 . 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 rAAV-based 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 transfection- and/or infection-based approaches using mammalian cells , or insect cells . 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 .
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 . 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 . 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 helper genes. The influence of different cultivation temperatures on Adenovirus replication kinetics and rAAV productivity in the transient packaging approaches were analyzed. Furthermore, we investigated differential gene expression in response to temperature downshifts.
Analysis of differential gene expression in HeLa triggered by different cultivation temperatures.
Differential expression at
Mode of regulation
Taken together, these first data provide the basis for a successful generation of temperature inducible stable producer cells carrying all genetic elements required for rAAV production. A versatile and high-titre rAAV production platform based on such cells will be applicable for industrial-scale manufacturing and thus has the potential to open AAV-based gene therapy to a high number of patients.
- Ars E, Serra E, de la Luna S, Estivill X, Lázaro C: Cold shock induces the insertion of a cryptic exon in the neurofibromatosis type 1 (NF1) mRNA. Nucl Acids Res. 2000, 28 (6): 1307-1312.PubMed CentralView ArticlePubMedGoogle Scholar
- Asokan A, Schaffer D, Samulski JR: The AAV Vector Toolkit: Poised at the Clinical Crossroads. Mol Ther. 2012, 20 (4): 699-708.PubMed CentralView ArticlePubMedGoogle Scholar
- Aucoin MG, Perrier M, Kamen AA: Critical assessment of current adeno-associated viral vector production and quantification methods. Biotechnol Adv. 2008, 26 (1): 73-88.View ArticlePubMedGoogle Scholar
- Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, Nitschko H, Busch U, Sing A, Ehrhardt A, Baiker A: Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeats. Hum Gene Ther Methods. 2012, 23 (1): 18-28.View ArticlePubMedGoogle Scholar
- Ayuso E, Mingozzi F, Bosch F: Production, purification and characterization of adeno-associated vectors. Curr Gene Ther. 2012, 10 (6): 423-436.View ArticleGoogle Scholar
- Bertran J, Moebius U, Hörer M, Rehberger B: Host cells for packaging a recombinant adeno-associated virus (RAAV), method for the production and use thereof. World Intellectual Property Organization. 2002, WO 02/20748 A2Google Scholar
- Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L, Conlon TJ, Boye SL, Flotte TR, Byrne BJ, Jacobson SG: Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther. 2008, 19: 979-990.PubMed CentralView ArticlePubMedGoogle Scholar
- Manno CS, Chew AJ, Hutchison S, Larson PJ, Herzog RW, Arruda VR, Tai SJ, Ragni MV, Thompson A, Ozelo M, Couto LB, Leonard DG, Johnson FA, McClelland A, Scallan C, Skarsgard E, Flake AW, Kay MA, High KA, Glader B: AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood. 2003, 101 (8): 2963-2972.View ArticlePubMedGoogle Scholar
- Matsushita T, Okada T, Inaba T, Mizukami H, Ozawa K, Colosi P: The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production. J Gen Virol. 2004, 85 (8): 2209-2214.View ArticlePubMedGoogle Scholar
- Mingozzi F, High KA: Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet. 2011, 12 (5): 341-355.View ArticlePubMedGoogle Scholar
- Nishiyama H, Higashitsuji H, Yokoi H, Itoh K, Danno S, Matsuda T, Fujita J: Cloning and characterization of human CIRP (cold-inducible RNAbinding protein) cDNA and chromosomal assignment of the gene. Gene. 1997, 204: 115-120.View ArticlePubMedGoogle Scholar
- Sonna LA, Fujita J, Gaffin SL, Lilly CM: Molecular biology of thermoregulation invited review: Effects of heat and cold stress on mammalian gene expression. J Appl Physiol. 2002, 92: 1725-1742.View ArticlePubMedGoogle Scholar
- Sonntag F, Schmidt K, Kleinschmidt JA: A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci USA. 2010, 107 (22): 10220-10225.PubMed CentralView ArticlePubMedGoogle Scholar
- Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC: Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol Bioeng. 2004, 88 (6): 707-721.View ArticlePubMedGoogle Scholar
- UniQure BV: [http://www.uniqure.com/news/167/189/uniQure-s-Glybera-First-Gene-Therapy-Approved-by-European-Commission.html]
- Urabe M, Ding C, Kotin RM: Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gen Ther. 2002, 13: 1935-1943.View ArticleGoogle Scholar
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