Minicircle DNA vectors for gene therapy: advances and applications

Abstract

Achieving in-target and persistent transgene expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.

Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site

“…In contrast, non-viral gene delivery methods have suffered from low transfection efficiencies and short sustainability despite many efforts to develop an efficient technology as a clinically available tool. In this study, we focused on a minicircle for non-viral gene delivery in MSCs because minicircle has shown great promise in various types of cells with greatly enhanced transfection efficiency and prolonged transgene expression [11,12]. Initially, we used the conventional cationic liposome method for MSC transfection with minicircles, but we found that the transfection efficiency was disappointingly low (<5%) (Fig.…”

“…(E) Decreases of wound area were represented after wounding, expressed as percentage of the initial wound size at 0 day: MC-LG (circle) and MC-LG þ ME-C4 (square). The data are shown as mean ± standard deviation values from n ¼ 3, *p < 0.05, **p < 0.01. rarely integrated into host genome as an episomal vector [12].…”

“…Besides the high transfection efficiency, minicircles have an advantage of sustained expression, which lasts several weeks after the transfection [11,12]. We directly analyzed the levels of transgene expressions in hAT-and hBM-MSCs by Western blot and fluorescence microscopic images for seven days after microporation (Fig.…”

“…The plasmids devoid of CpG dinucleotide sequences have been shown to generate higher and more sustainable expression of transgenes [9,10]. Among such efforts, minicircles have been highlighted as minimized eukaryotic expression cassettes that are devoid of bacterial plasmid backbone containing many CpG motifs [11,12]. They are usually generated by site-specific recombination to remove bacterial backbone sequences after the propagation of parental plasmid [13e16].…”

“…Especially, genetically engineered Escherichia coli capable of inducible in vivo recombination and following the enzymatic degradation of bacterial backbone made a great improvement in minicircle production by reducing time and labor to similar levels as those of routine plasmid DNA preparation [16]. Minicircles have been employed for various purposes including transgene expression, gene therapy, DNA vaccine, and the generation of induced pluripotent stem cells [11,12,17]; however, there have been only a few studies that used minicircles for transfection of MSCs [18,19]. In these studies, minicircles were delivered to MSCs by the conventional cationic liposome method or nucleofection technology, but the resulting transfection efficiencies were low (less than 35%).…”