Journal article
Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers
Human gene therapy, Vol.26(6), pp.334-346
06/01/2015
DOI: 10.1089/hum.2015.001
PMCID: PMC4492606
PMID: 25763813
Abstract
The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (
CFTR
) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air–liquid interface (ALI), as well as in the ferret airway
in vivo
, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the
CFTR
expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRΔR). This vector yielded an ∼17-fold increase in expression of CFTR and significantly improved Cl
–
currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway.
Details
- Title: Subtitle
- Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers
- Creators
- Ziying Yan - 2Center for Gene Therapy, University of Iowa School of Medicine, Iowa City, IA 52242Xingshen Sun - 1Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, IA 52242Zehua Feng - 1Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, IA 52242Guiying Li - 3Department of Surgery, University of Iowa School of Medicine, Iowa City, IA 52242John T Fisher - 1Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, IA 52242Zoe A Stewart - 3Department of Surgery, University of Iowa School of Medicine, Iowa City, IA 52242John F Engelhardt - 4Department of Internal Medicine, University of Iowa School of Medicine, Iowa City, IA 52242
- Resource Type
- Journal article
- Publication Details
- Human gene therapy, Vol.26(6), pp.334-346
- DOI
- 10.1089/hum.2015.001
- PMID
- 25763813
- PMCID
- PMC4492606
- NLM abbreviation
- Hum Gene Ther
- ISSN
- 1043-0342
- eISSN
- 1557-7422
- Publisher
- Mary Ann Liebert, Inc
- Language
- English
- Date published
- 06/01/2015
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Surgery; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025412602771
Metrics
16 Record Views