Minicircle DNA Vectors Achieve Sustained Expression Reflected by Active Chromatin and Transcriptional Level

Abstract

Current efforts in nonviral gene therapy are plagued by a pervasive difficulty in sustaining therapeutic levels of delivered transgenes. Minicircles (plasmid derivatives with the same expression cassette but lacking a bacterial backbone) show sustained expression and hold promise for therapeutic use where persistent transgene expression is required. To characterize the widely-observed silencing process affecting expression of foreign DNA in mammals, we used a system in which mouse liver presented with either plasmid or minicircle consistently silences plasmid but not minicircle expression. We found that preferential silencing of plasmid DNA occurs at a nuclear stage that precedes transport of mRNA to the cytoplasm, evident from a consistent >25-fold minicircle/plasmid transcript difference observed in both nuclear and total RNA. Among possible mechanisms of nuclear silencing, our data favor chromatin-linked transcriptional blockage rather than targeted degradation, aberrant processing, or compromised mRNA transport. In particular, we observe dramatic enrichment of H3K27 trimethylation on plasmid sequences. Also, it appears that Pol II can engage the modified plasmid chromatin, potentially in a manner that is not productive in the synthesis of high levels of new transcript. We outline a scenario in which sustained differences at the chromatin level cooperate to determine the activity of foreign DNA.

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

“…These minimal expression cassettes are derived from 'parental' plasmid (pp) DNA, through site-specific recombination (induced via L-arabinose operon system). Their use results in sustained transgene expression due to lower activation of nuclear transgene silencing mechanisms, and reduced immunogenic responses [30]; the latter resulting in higher safety. The clinical potential of mCs has been exploited for non-viral DNA reprogramming to generate human induced pluripotent stem cells [31].…”

“…Guidance on the use of medicinal products for gene transfer advise great care in the use of selection markers used in such products due to their significant potential to impact human therapy. This is especially pertinent to the presence of antibiotic resistance genes since these can restrict more general treatment options [30,46,56,57]. Indeed, medical and clinical regulatory bodies advise limiting the use of antibiotic resistance genes for therapy where feasible [58].…”

“…20% at 10 days post-transfection, but longer time points were not studied [32]. It is possible that the prolonged gene expression with mC DNA versus conventional plasmids is due to the lack of transcriptional silencing associated with the presence of unmethylated CpG islands [30,46]. The removal of these individual sequences using conventional molecular biology cloning techniques is laborious and expensive; in comparison the mC DNA technology enables a facile, cheaper approach for eliminating bacterial sequences, involving fewer and less time consuming procedures.…”

Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology

“…[28] We and others have reasoned that the superior transfection efficiency is likely due to mC engineered cells containing higher transgene copies per cell versus conventional plasmids. The mC vectors are also compliant with regulations on the use of medicinal products for gene transfer which restrict the use of selection markers due to their substantial potential to impact therapeutic outcomes [29][30][31][32][33]. Despite the obvious clinical translational benefits offered by this genetic modification methodology for regenerative neurology, the feasibility of mC based delivery of therapeutic genes to neural transplant populations has never been evaluated.…”

Production of Double-stranded DNA Ministrings

“…As they are solely comprised of the eukaryotic expression cassette, DNA minivectors, such as DNA minicircles 3 , are a better alternative for gene delivery as they exhibit improved extracellular and intracellular bioavailability and improved gene expression due to their reduced size and absence of immunostimulatory prokaryotic elements 4 . The reduced vector size fares better with respect to resistance to shear forces associated with in vivo administration to a target site 5 .…”