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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.

SUBMITTER: Stenler S

PROVIDER: S-EPMC3910003 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression.

Stenler Sofia S Wiklander Oscar Pb OP Badal-Tejedor Maria M Turunen Janne J Nordin Joel Z JZ Hallengärd David D Wahren Britta B Andaloussi Samir El SE Rutland Mark W MW Smith C I Edvard CI Lundin Karin E KE Blomberg Pontus P

Molecular therapy. Nucleic acids 20140107

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 ...[more]

PMID: 24399204

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Project description:To overcome the problems with current mineral supplements for laying hens including low absorption, mineral antagonism, and high cost, we developed mineral element fermentation complexes (MEFC) by synergistically fermenting bean dregs and soybean meal with strains and proteases and complexing with mineral elements. The fermentation complexation process was optimized based on the small peptide and organic acid contents and the complexation rate of mineral elements after fermentation. The optimal conditions were as follows: the total inoculum size was 5% (v/w), 15% (w/w) wheat flour middling was added to the medium, and mineral elements (with 4% CaCO3) were added after the completion of aerobic fermentation, fermentation at 34°C and 11 days of fermentation. Under these conditions, the complexation rates of Ca, Fe, Cu, Mn, and Zn were 90.62, 97.24, 73.33, 94.64, and 95.93%, respectively. The small peptide, free amino acid, and organic acid contents were 41.62%, 48.09 and 183.53 mg/g, respectively. After 60 days of fermentation, 82.11% of the Fe in the MEFC was ferrous ions, indicating that fermentation had a good antioxidant effect on ferrous ion, and the antioxidant protection period was at least 60 days. Fourier transform infrared spectroscopy showed that the mineral ions were complexed with amino and carboxyl groups. The added mineral elements promoted microbial growth, protein degradation, and organic acid secretion and significantly improved fermentation efficiency. Animal experiments showed that MEFC had positive effects on several parameters, including production performance (average daily feed intake, P < 0.05; egg production rate, P < 0.05; and average egg weight, P < 0.05), mineral absorption, intestinal morphology (villus height to crypt depth ratio in the jejunum and ileum, P < 0.05), and blood routine and biochemical indexes (red blood cells, P < 0.05; hemoglobin, P < 0.05). This study provides theoretical support for the development of mineral complexes for laying hens via fermentation.

Dataset Information

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression.

Publications

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression.

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