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

Abstract

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.

Mini-intronic plasmid vaccination elicits tolerant LAG3+CD8+T cells and inferior antitumor responses

“…These data correspond to previous studies showing that silencing of gene expression from conventional plasmid vectors is more pronounced in quiescent tissue in vivo. 19,34,35 Given the similarity of gene expression from minicircle or MIP vectors, and given the ease of preparation of pure MIP vectors without contaminating DNA populations common with minicircle preparations (Fig. S2), subsequent studies focused on the use of the MIP construct.…”

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

“…It should be noted that given their clinical utility, a novel method involving affinity-based chromatography has been developed for obtaining highly purified mC DNA for gene therapy and vaccination, enabling safe production at an industrial scale [33,62,63]. Together, these factors highlight the critical advantages offered by mC DNA vector technology in conjunction with magnetofection, for clinical translational applications involving genetically engineered neural transplant cells (summarized in Fig.…”

A non‐viral gene therapy for treatment of recessive dystrophic epidermolysis bullosa

“…It is theorized that MC DNA may even effect a larger area in vivo than the standard plasmid gene therapy (9). The increased diffusion coefficient through tissue of small particles such as HPAE-MC7 is very attractive for dermatological conditions like RDEB.…”