Optimization of the piggyBac transposon using mRNA and insulators: toward a more reliable gene delivery system

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expr...

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Bibliographic Details
Published in:PloS one 2013-12, Vol.8 (12), p.e82559-e82559
Main Authors: Bire, Solenne, Ley, Déborah, Casteret, Sophie, Mermod, Nicolas, Bigot, Yves, Rouleux-Bonnin, Florence
Format: Article
Language:English
Subjects:
Bioavailability
Biotechnology
Blotting, Western
Chromosomes
Deoxyribonucleic acid
DNA
Electric insulators
Epigenetics
Expression vectors
Fibroblasts
Gene expression
Gene therapy
Gene transfer
Gene Transfer Techniques
Genetic Vectors - genetics
Genomes
Genotoxicity
HeLa Cells
Humans
Insulators
Integration
Life Sciences
Mammals
Messenger RNA
Optimization
Other
Plasmids
Regulatory sequences
Ribonucleic acid
RNA
RNA, Messenger - genetics
Side effects
Transposase
Transposases - genetics
Transposases - metabolism
Transposition
Transposons
Vectors (Biology)
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Summary:Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0082559