Journal article
Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes
Molecular therapy. Methods & clinical development, Vol.11, pp.40-51
12/14/2018
DOI: 10.1016/j.omtm.2018.09.005
PMCID: PMC6205362
PMID: 30397626
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.
Details
- Title: Subtitle
- Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes
- Creators
- Zekun Wang - Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USAFang Cheng - Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USAJohn F Engelhardt - Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USAZiying Yan - Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USAJianming Qiu - Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA
- Resource Type
- Journal article
- Publication Details
- Molecular therapy. Methods & clinical development, Vol.11, pp.40-51
- DOI
- 10.1016/j.omtm.2018.09.005
- PMID
- 30397626
- PMCID
- PMC6205362
- NLM abbreviation
- Mol Ther Methods Clin Dev
- ISSN
- 2329-0501
- eISSN
- 2329-0501
- Publisher
- American Society of Gene & Cell Therapy
- Grant note
- DOI: 10.13039/100000060, name: NIAID; DOI: 10.13039/100000002, name: NIH, award: YAN15XX0; DOI: 10.13039/100000897, name: Cystic Fibrosis Foundation
- Language
- English
- Date published
- 12/14/2018
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025307202771
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