Performance of High Quality Minicircle DNA for In Vitro and In Vivo Gene Transfer

Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany, .
Molecular Biotechnology (Impact Factor: 1.88). 04/2012; 53(1). DOI: 10.1007/s12033-012-9535-6
Source: PubMed


Plasmid DNA is frequently used particularly for nonviral gene therapy. Conventional plasmid DNA contains bacterial backbone and resistance gene sequences, as well as immunogenic CpG motifs. These components are not required for transgene expression. They represent a potential risk for safe clinical application and reduce gene transfer rates as well as transgene expression. To overcome these drawbacks, the minicircle technology is removing such sequences, to improve performance and also to reduce DNA size. Here, we show the effective production of luciferase, GFP, or lacZ-carrying minicircle DNA with high yield and reproducible high quality. They are used for lipofection or electroporation gene transfer into human melanoma and colon carcinoma cell lines. Comparison of respective parental plasmid and minicircle-mediated luciferase gene transfer shows improved luciferase expression by minicircle in all cell lines. This is not associated with increase in intracellular minicircle copy numbers after lipofection or electroporation. The minicircles rather mediate enhanced transgene mRNA transcription compared to their parental plasmids. In addition, FACS analysis revealed increase in counts of GFP positive cells after minicircle gene transfer, indicating higher gene transfer rates. Furthermore, minicircle showed also improved performance in vivo after jet-injection gene transfer. Therefore, availability of minicircles with reproducible high quality and sufficient amount makes them an applicable and effective alternative to conventional plasmid gene vectors.

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    • "These unique characteristics associated to their smaller size, significantly increases their safety and in vivo efficacy [15]. This enhanced biological performance has been recently evidenced by Kobelt and co-workers which reported that in vivo administration of mcDNA induces higher levels of transgene expression in comparison to plasmid-based vectors [16]. Despite these improvements in both nanocarriers and DNA vectors, it has been demonstrated that imprinting stimuli-responsive properties in nanoformulations designed for co-delivery may provide an added biological efficacy since a more controlled biodegradation and sitespecific , on-demand release can be promoted [17]. "
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    ABSTRACT: The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock co-polymer micelles based on poly(2-ethyl-2-oxazoline)-poly(l-lactide) grafted with bioreducible polyethylenimine (PEOz-PLA-g-PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8days following mcDNA-micelles intratumoral administration. Furthermore, drug-gene co-delivery in PEOz-PLA-g-PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy. Copyright © 2015. Published by Elsevier B.V.
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    ABSTRACT: Introduction Overview of Magnetofection Principles Cellular Uptake Diffusion through the Cytoplasm Transgene Expression Conclusions References
    No preview · Chapter · Apr 2013
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    ABSTRACT: This chapter contains sections titled: Introduction Production of Minicircles Analytics of Minicircle Production Future Goals Acknowledgments References
    No preview · Chapter · Apr 2013
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