Dataset Information

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes.

ABSTRACT: Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, and (3) pAAVRepCap supports rAAV production in HEK293 cells. Despite a production yield of 1-2 log lower than that using pAdHelper (expressing Ad genes E2A, E4, and VA), rAAV vector produced using pBocaHelper transduced cells as efficiently as that produced using pAdHelper. The low vector production is largely due to the inefficient expression of the AAV Rep52 and capsid proteins, as well as reduced rAAV genome replication. When the AAV capsid proteins and Rep52 were ectopically expressed under strong promoters, the enhanced protein expression significantly improved the rAAV production using pBocaHelper, approaching a level of 50%-70% of that produced using pAdHelper. Through further dissection of the helper functions from pAdHelper in a five-plasmid transfection system, we found that the addition of the Ad E2A gene to the above HBoV1 helper system significantly increased rAAV DNA replication, which increased the rAAV vector production to a level of 3-7 times higher than that using pAdHelper. We finally combined HBoV1 NP1 and NS2 genes with Ad helper genes to create a novel dual helper plasmid (pABHelper) for rAAV vector production in the conventional three-plasmid transfection system. The pABHelper facilitated rAAV production at a yield ?2 times higher than that using the pAdHelper.

SUBMITTER: Wang Z

PROVIDER: S-EPMC6205362 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes.

Wang Zekun Z Cheng Fang F Engelhardt John F JF Yan Ziying Z Qiu Jianming J

Molecular therapy. Methods & clinical development 20181004

Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, ...[more]

PMID: 30397626

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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.

Project description: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.

Dataset Information

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes.

Publications

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes.

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