Minicircle Research Articles

Histone Modifications are Associated with the Persistence or Silencing of Vector-mediated Transgene Expression In Vivo

One of the major obstacles to success in non-viral gene therapy is transcriptional silencing of the DNA vector. The mechanisms underlying gene silencing/repression in mammalian cells are complex and remain unclear. Because changes in chromatin structure and, in particular, histone modifications are involved in transcriptional regulation of endogenous genes, we hypothesized that changes in the pattern of histone modifications were related to the observed transcriptional silencing of exogenous DNA vectors. We used antibodies against specific modified histones to perform chromatin immunoprecipitation (ChIP) analyses on liver lysates from mice transfected with two types of plasmids: (i) DNA minicircles (MCs) devoid of bacterial plasmid backbone DNA, which showed marked persistence of transgene expression, and (ii) their parental plasmids, which were silenced over time. Silencing of the transgene from the parental vectors was accompanied by an increase in heterochromatin-associated histone modifications and a decrease in modifications typically associated with euchromatin. Conversely, the pattern of histone modifications on the MC DNA was consistent with euchromatin. Our data indicates that (i) episomal vectors undergo chromatinization in vivo, and (ii) both persistence and silencing of transgene expression are associated with specific histone modifications.

1068. Plasmid DNA Sequences Present in Conventional HSV Amplicon Vectors Cause and Rapid Transgene Silencing by Inactive Chromatin Formation

The HSV-based amplicon vector, a bacteria-virus-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle amplicon). Although the same dose of the minicircle (MC) amplicon vector in normal human fibroblasts resulted in expression of luciferase approximately 20 times higher than that produced by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Similar results were obtained with nondividing normal human astrocytes in culture, precluding the possibility that this phenomenon is fibroblast specific. Finally, nude mice injected with MC amplicon exhibited higher and more sustained expression of luciferase than those injected with conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. These findings are a significant first step towards developing a novel herpes simplex virus (HSV) amplicon vector that can achieve enhanced long-term transgene expression.

Unusual organisation of the plastid genome of the dinoflagellate Amphidinium operculatum

We have previously reported an anomalous organization of several plastid genes in the dinoflagellate Amphidinium operculatum. These genes are located on individual minicircles of around 2.4 kbp, with a single gene on each minicircle. Each minicircle also has a `core? region, which is similar but not identical between minicircles. This gene organization contrasts markedly with that in other plastid genomes, which typically have 100 or so genes located on one molecule. Using dinoflagellate DNA and PCR with primers based on conserved regions of several genes, we have identified additional genes for plastid proteins on minicircles. We have identified one minicircle containing two genes. PCR using primers based on the core region has allowed us to identify a number of `empty? minicircles that do not appear to contain any genes (although one empty minicircle has short regions of similarity to other genes). These empty minicircles have a recognisable core region, however. We have carried out transcript analysis of a number of minicircles, including the one carrying two genes. We have also tested the hypothesis that dinoflagellate plastid transcripts undergo editing.

The evolution of minicircles and guide RNAs in Trypanosomes

The evolution of minicircles and guide RNAs in Trypanosomes