title: Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression.
abstract: The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.Molecular Therapy-Nucleic Acids (2014); doi:10.1038/mtna.2013.67.
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sofia
authors.initials: S
authors.lastname: Stenler
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Oscar Pb
authors.initials: OP
authors.lastname: Wiklander
authors.affiliation: YKI, Institute for Surface Chemistry, Stockholm, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Badal-Tejedor
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Janne
authors.initials: J
authors.lastname: Turunen
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Joel Z
authors.initials: JZ
authors.lastname: Nordin
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: Hallengärd
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Britta
authors.initials: B
authors.lastname: Wahren
authors.affiliation: 1] Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden [2] Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Samir El
authors.initials: SE
authors.lastname: Andaloussi
authors.affiliation: Department of Chemistry, Surface Chemistry, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Rutland
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
authors.forename: C I Edvard
authors.initials: CI
authors.lastname: Smith
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Karin E
authors.initials: KE
authors.lastname: Lundin
authors.affiliation: 1] Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden [2] Vecura, Clinical Research Center, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Pontus
authors.initials: P
authors.lastname: Blomberg