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Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

ABSTRACT: Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

SUBMITTER: Satkunanathan S

PROVIDER: S-EPMC4236031 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

Satkunanathan Stifani S Wheeler Jun J Thorpe Robin R Zhao Yuan Y

Human gene therapy 20140911 11

Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, var ...[more]

PMID: 25072415

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Project description:The genome of recombinant adeno-associated virus 2 (rAAV2) remains a promising candidate for gene therapy for cystic fibrosis (CF) lung disease, but due to limitations in the packaging capacity and the tropism of this virus with respect to the airways, strategies have evolved for packaging an rAAV2 genome (up to 5.8 kb) into the capsid of human bocavirus 1 (HBoV1) to produce a chimeric rAAV2/HBoV1 vector. Although a replication-incompetent HBoV1 genome has been established as a trans helper for capsid complementation, this system remains suboptimal with respect to virion yield. Here, a streamlined production system is described based on knowledge of the involvement of HBoV1 nonstructural (NS) proteins NS1, NS2, NS3, NS4, and NP1 in the process of virion production. The analyses reveal that NS1 and NS2 negatively impact virion production, NP1 is required to prevent premature termination of transcription of the cap mRNA from the native genome, and silent mutations within the polyadenylation sites of the cap coding sequence can eliminate this requirement for NP1. It is further shown that preventing the expression of all NS proteins significantly increases virion yield. Whereas the expression of capsid proteins VP1, VP2, and VP3 from a codon-optimized cap mRNA was highly efficient, optimal virion assembly, and thus potency, required enhanced VP1 expression, entailing a separate VP1 expression cassette. The final NS protein-free production system uses three-plasmid co-transfection of HEK293 cells, with one trans helper plasmid encoding VP1 and the AAV2 Rep proteins, and another encoding VP2-3 and components from adenovirus. This system yielded >16-fold more virions than the prototypic system, without reducing transduction potency. This increase in virion production is expected to facilitate greatly both research on the biology of rAAV2/HBoV1 and preclinical studies testing the effectiveness of this vector for gene therapy of CF lung disease in large animal models.

Dataset Information

Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

Publications

Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

Similar Datasets

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