Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer

Abstract

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.

Host cell in vivo production of the synthetic drug anti–CD25/IL‐10 using minicircle vector

“…MCs are a class of enhanced nonviral DNA vectors and are almost entirely composed of the transgene expression cassette. Free of the plasmid backbone DNA, the MC DNA vector has minimal detrimental effects and excellent transgene-expressing profiles (7,11,23). Importantly, technology is available to produce abundant, high-quality MCs at only a small fraction of the cost of producing targets (8,11).…”

Engineering DNA vaccines against infectious diseases

“…It has been reported that the FDA recommends integration studies, regardless of the delivery method, if there are greater than 10,000 copies of foreign DNA per microgram of host DNA [71]. Interestingly, minicircle DNA [51,53,54,72,73] and ministring DNA [74,75] have been reported to have less risk of insertional mutagenesis because the major bacterial DNA (i.e., the unmethylated CpG repeats functioning as PAMPs) is removed.…”

“…This large-scale pDNA production is cost-effective in comparison to LAV and IV manufacturing processes, possibly making the technology cheaper in the long run for consumers. The relatively recent development of minicircles (episomal DNA)/miniplasmids (i.e., P1, P7, and F)/ministrings [50][51][52][53][54][55] can potentially be transported more easily through cellular barriers to improve bioavailability [51].…”

Impact of gene editing on the study of cystic fibrosis

“…The repeated delivery of plasmids expressing the CFTR cDNA to stablise lung function in patients (Alton et al 2015) suggests that plasmids of a similar size could be used to deliver gene-editing nucleases, and clinical trials to assess the safety and efficacy of direct RNA-editing delivered by non-viral vectors are underway (ProQR clinical trials). Nanocomplex formulation of lipids and peptides to efficiently deliver minicircle DNA-encoding reporter genes to mouse lung also provides proof of principle for delivery of the cDNAs encoding gene-editing nucleases (Munye et al 2016). Adeno-associated virus (AAV) has been used to deliver cDNA encoding ZFNs (Li et al 2011) and Cas9 derived from S. aureus (Ran et al 2015), and adenovirus has been used to deliver cDNA encoding TALENs (Holkers et al 2014).…”