Journal article
Establishment of a High-Yield Recombinant Adeno-Associated Virus/Human Bocavirus Vector Production System Independent of Bocavirus Nonstructural Proteins
Human gene therapy, Vol.30(5), pp.556-570
05/01/2019
DOI: 10.1089/hum.2018.173
PMCID: PMC6534084
PMID: 30398383
Abstract
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.
Details
- Title: Subtitle
- Establishment of a High-Yield Recombinant Adeno-Associated Virus/Human Bocavirus Vector Production System Independent of Bocavirus Nonstructural Proteins
- Creators
- Ziying Yan - 2Center for Gene Therapy, University of Iowa, Iowa City, IowaWei Zou - 3Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KansasZehua Feng - 1Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IowaWeiran Shen - 3Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KansasSoo Yeun Park - 1Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IowaXuefeng Deng - 3Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KansasJianming Qiu - 3Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KansasJohn F Engelhardt - 2Center for Gene Therapy, University of Iowa, Iowa City, Iowa
- Resource Type
- Journal article
- Publication Details
- Human gene therapy, Vol.30(5), pp.556-570
- DOI
- 10.1089/hum.2018.173
- PMID
- 30398383
- PMCID
- PMC6534084
- NLM abbreviation
- Hum Gene Ther
- ISSN
- 1043-0342
- eISSN
- 1557-7422
- Publisher
- Mary Ann Liebert, Inc., publishers
- Language
- English
- Date published
- 05/01/2019
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025366202771
Metrics
16 Record Views